In vitro studies investigated the cellular effects of Aβ in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aβ-induced focal AMD-like pathology within 2 weeks. Aβ exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aβ co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aβ within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aβ were identified as deficient in cargo-carrying lysosomes at time points that are critical to POS degradation. These findings imply that Aβ accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes.Alzheimer’s disease-associated amyloid beta (Aβ) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aβ-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aβ The aggregation of intracellular proteins/lipids in the senescent retina is associated with the common irreversible blinding condition, age-related macular degeneration (AMD). These aggregates arise from the incomplete degradation of photoreceptor outer segments (POS) and the accumulation of intracellular lipofuscin within retinal pigment epithelial (RPE) cells ,2. The b1-42 was subretinally injected into wild-type mouse eyes to recapitulate the high Aβ burden in AMD retinas. The development of AMD-like pathology was longitudinally evaluated in living eyes using non-invasive techniques including color fundus photography (CFP), full-field electroretinograms (ERG) and optical coherence tomography (OCT). These studies were complemented with end-point histological analysis of mouse eyes. Pathology in affected tissue layers was investigated further using in vitro models of choroidal endothelial and retinal pigment epithelial cells. The intracellular fate of Aβ was studied in detail as it was rapidly internalized by RPE cells. Our findings reveal that impaired proteolytic mechanisms in RPE cells coupled to the rapid accumulation of Aβ in lysosomes could be a novel pathway of cellular dysfunction. Next, we investigated potential effects of Aβ-mediated pathology related to the cells’ ability to traffic photoreceptor outer segments, which are processed daily by the RPE. Our findings not only provide evidence of how Aβ targets specific tissues in the outer retina, but also reveal altogether novel and unexpected mechanisms through which pathology can be initiated at the cellular level, contributing to sight loss in later life.To address these questions, we exploited an in vivo model in which human oligomeric Aβ1-42 was prepared according to previously published methods [1-42  was reconstituted to 1 mg/mL in HFIP, vortexed for 60 s and sonicated at 50 Hz for 5 min. The resuspension solvent was subsequently removed with O2-free dry nitrogen and vacuum desiccation for 30 min. Resultant peptidic films were then suspended in DMSO to a concentration of 1 mg/mL, vortexed and applied along with 40 μL Aβ buffer stacker to a 2 mL, 7K Zeba buffer exchange column pre-equilibrated with Aβ buffer . Columns were centrifuged at 1000× g for 2 min at 4 °C to facilitate buffer exchange and eluates spun at 16,000× g for 30 min to remove preformed fibrillar aggregates. Aβ1-42 concentrations were determined with Beer–Lambert’s Law using the absorbance measured at A280 with a ND-1000 Nano drop spectrophotometer and the molar extinction co-efficient of Aβ (1490 M−1·cm−1). Alexa Fluor®-tagged Aβ was prepared as described. However, prior to application of Aβ1-42 to the exchange columns, 20 μL sodium bicarbonate and 10 μL of 11.3 nm/μL dye dissolved in ddH2O were added to Aβ1-42 in DMSO and incubated for 15 min at room temperature. Concentrations of Alexa Fluor 488 or 647-tagged Aβ1-42 were determined as described above taking into account the respective Alexa Fluor contributions to 280 nm absorbance as stated in the manufacturer’s protocol. Preparations within the range of 50–130 μM were incubated on ice for 1.5 h prior to use in experiments to ensure the highest concentration of oligomeric Aβ1-42. Bacterial endotoxin levels in preparations were measured using a Limulus Amebocyte Lysate (LAL) Endosafe PTS cartridge , which demonstrated that <0.5 EU/mL was present in samples. This is in line with the limits specified by the US Food and Drug Administration (FDA).Oligomeric Aβ methods ,23. Briead libitum. Conventional cages contained Lignocel 2/2  bedding and environmental enrichment, housing no more than 10 mice per cage. All experiments were performed between 9:00 and 18:00 h in the light stage of the light–dark cycle. Animal body weight was monitored throughout experiments as a surrogate measure of welfare and studies terminated if a ˃10% change occurred over a period of 3 days. The sample size was determined using the resource equation method taking into account attrition rates after successful subretinal injections. Mice were euthanized on day 15 post-transscleral subretinal injection by cervical dislocation and cutting of the carotid artery.Animal studies were overseen by the institutions’ Ethical Research Committee and were carried out in accordance with the UK Animal (Scientific Procedures) Act of 1986. Experiments also conformed to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Mice were obtained from the Biomedical Research Facility . For non-invasive retinal scans, 13 female test-naïve C57BL/6J mice were used aged 117 ± 4 days and weighing 22.8 ± 0.23 g. Animals were housed in a 12/12 h light–dark cycle at 19–24 °C and allowed access to standard laboratory chow and water w/v phenylephrine hydrochloride  for 2 min followed by 1 drop of 1% w/v Tropicamide  for 2 min. Dilated eyes were kept hydrated throughout anesthesia via repeated application of Viscotears . During this period, mice were also maintained on a heat pad and their breathing rate was monitored. Animals were revived via subcutaneous injection with 200 μL 0.5 mg/mL antipamezole hydrochloride , recovered on a heat pad and returned to the home cage once righting reflexes had returned.Mice were anaesthetized with 1 mg ketamine  and 0.005 mg dexmedetomine hydrochloride  per 10 g weight via intraperitoneal (IP) injection. Ketamine facilitates pupillary dilation and is thus an optimal anesthetic for ocular imaging and functional assessments. To minimize stress, animals were subsequently housed in an environment with minimal lighting until cessation of motor function. Pupils were dilated by application of 1 drop 2.5% 1-42 (n = 7) or vehicle (n = 6) was then slowly injected by controlled depression of the plunger. Needles were left in place for 5–10 s afterwards to prevent potential back flow of any fluid. The success of the subretinal injection was confirmed by observing presence of a retinal bleb through the operating microscope. A co-investigator was on hand at all times to refocus the microscope, assist with stabilizing the mouse and visually verify subretinal injection via the second eyepiece on the operating microscope. Both eyes of each animal was subretinally injected with Aβ or vehicle in this manner. Animals with retinal bleeds or perforation of the retina (indicated by a bolus in the vitreous) were excluded from studies. Treatment groups were assigned randomly where the statistical unit was the individual mouse.Transscleral subretinal injections were carried out via the choroid and BrM without retinal penetration using a Stativ Opmi CS/S4 surgical microscope . For a visual presentation of transscleral subretinal injection surgery, we refer the reader to Dumitrescu et al. . This meCFP of mouse retinas were obtained using the Micron III Retinal Imaging System . Mice were placed on the imaging platform with corneas aligned with the lens. Images were acquired using a brightfield imaging modality (450–650 nm) and visualized using Micron III Retinal Imaging Microscope Software . In the case of re-imaging retinas at 8 days post-subretinal injection, the optic nerve and retinal veins were used as a reference points to locate the injection site seen immediately post-injection as a retinal ‘bleb’. Further, the entire retina was assessed with the microscope and the lesion area captured to ascertain the full extent of induced pathology.2) of 1.5 mm diameter for 1 ms. Stimulation was performed in two sweeps with a two minute interval from which an average recording was determined. In all cases, oculus dexter measurements were carried out first. Animals were assessed randomly. ERGs were visualized in the V3 Phoenix LabScribe ERG software suite . A-wave and B-wave amplitudes were calculated as the measurement from baseline to the A-wave trough and the A-wave trough to the B-wave peak, respectively. The implicit time  for both the A and B waves was also recorded.Mice were dark adapted for 12 h prior to ERG recordings and maintained at 27 °C throughout the duration of the procedure. ERG traces were recorded using the Generation II Image-Guided ERG modality attachment to the Micron III Retinal Imaging System  which was housed inside a 6-panel aluminum copper mesh Faraday cage  to minimize potential electrical interference. Animals were placed on a heated platform and were connected to three electrodes as follows: (1) a ground electrode (inserted into the tail), (2) a reference electrode that was attached to the head, and (3) an electrode with corneal contact, which was achieved by positioning the cornea onto the gold-plated objective lens. ERGs were recorded by stimulation with white LED light . Mice were wrapped in a surgical swab, loaded into the imaging platform and the eye of interest positioned in line with the mouse retina lens using the multiaxial rodent alignment stage apparatus. The cornea was cleaned with surgical spears and Systane lubricant eye drops . Cross-sectional previews as well as real-time en face fundus images were used for final alignment with the OCT probe in the InVivoVue Clinic software . Images were centered round the optic nerve head before 1.4 mm volumetric scans were acquired through a 50 degree field of view comprising 100 B scans, each of which consisted of 1000 A scans. The whole retina scan function was first employed to confirm that the captured region encompassed the lesions and site of subretinal injection. This was evident in both en face and cross-sectional previews of Aβ-injected mice, but less so in vehicle-injected mice despite scanning the entire retina see . FinallyMice were enucleated at one week post-injection and eyes fixed in 4% PFA for 30 min at 4 °C, washed in 1xPBS and dehydrated through a series of sucrose gradients as follows: 5% (60 min), 10% (30 min), 12.5% (30 min), 15% (30 min) and 20% (overnight). Posterior ocular sections comprising the sclera, RPE/choroid and retina were subsequently embedded in optimal cutting temperature (OCT) medium and sectioned at 16 μm sections for hematoxylin and eosin staining (H&E). Slides were dried for 1 h and incubated with Gill’s III hematoxylin for 10 min, tap water for 8 min, 0.03% acid alcohol for 8 min, tap water for 8 min, 0.5% eosin for 1 min, distilled water (30 s) followed by incubation in 50%, 70%, 90% and 100% EtOH with 30 s at each step. Slides were placed in Xylene for 5 min and mounted with DPX mounting medium. Imaging were acquired using an Olympus dotSlide virtual microscopy system  and visualized using the OlyVIA software suite . Tissues were assessed within a 100 μm × 300 μm region of interest at 200 μm intervals and a binary score of 1 or 0 assigned to inner segments (IS), outer segments (OS) and RPE/Choroid to indicate retinal pathology. A total of 15 sections were analyzed per eye from 5 slides at 240 μm intervals (3 sections/slide). Tissues within a 200 μm radius from the injection site was excluded to prevent any effects of mechanical injury caused by the subretinal injection being included in the analysis. This was identified as a ‘knick’ in the tissue extending through the sclera, choroid and RPE, with evidence of more trauma than surrounding regions. This could also be seen as a discontinuity, or break in pigmented retinal layers along with damage to the outer neural retina.2. Cells were passaged at a ratio of 1:2 with a complete media change every 2–3 days. Cells cultured on 24 mm, 0.4 μm PET Transwell® Permeable Supports were maintained in 2 mL apical and 3 mL basal media volume with a 100% and 20% media change every 2–3 days, respectively [ARPE-19 cells in passages 23–26 (ATCC) were cultured in Dulbecco’s modified Eagle medium with 4.5 g/L L-D glucose, L-glutamine and pyruvate  supplemented with 1% heat inactivated FCS  and 100 units/mL penicillin, 0.1 mg/mL streptomycin  in a 37 °C humidified incubator with an atmosphere of 5% COectively ,26. RF/65 in 13 mm glass coverslips and cultured until 80% confluence (~5 days). A scratch was created at the center of coverslip using a sterile pipette. Reference points were etched to ensure that identical fields of view were imaged. Cells were washed with 1xHBSS to remove cellular debris, 1 μM Aβ1-42 or an equivalent volume of vehicle was applied and cells returned to the incubator for 0, 6, 24 or 48 h. Images were taken using the EVOS-XL Core imaging system across three biological replicates at x4 magnification. To account for any differences in scratch size between wells, data were normalized to the 0 h time point and converted into a percentage . Scratch closure was quantified by an investigator blinded to the identity of images using ImageJ  software. The cell migration rate (μm/h) was calculated using the formula Vmigration = |slope|/2xI [RF/6A choroidal endothelial cells (CECs) were seeded at 1.9 × 10ope|/2xI .4/well in 24 mm sized Transwell inserts and maintained for a minimum of 4 months prior to use. Secreted levels of human Vascular Endothelial Growth Factor (VEGF), Pigment Epithelium-Derived Factor (PEDF) and Aβ  were quantified in conditioned media from apical and basal compartments at 48 h after a complete media change (n = 3) using the following assays: Novex® human VEGF , human PEDF  and Aβ1-X . Assays were carried out according to the manufacturer’s instructions. Three technical replicates were assessed per biological replicate and protein concentrations determined by measuring the absorbance at 450 nm with a FLUOstar Optima microtiter plate reader .Cells were seeded at 5 × 101-42 was applied to 4 month old ARPE-19 cells cultured on 0.4 μm PET Transwells for either 2, 24 or 48 h. The cultures were washed in fresh medium and 500 μL of 1.7 mg/mL 40 KDa FITC-dextran was applied to the apical Transwell compartment. Then, 0.5, 1, 3 and 5 h after application, 100 μL of apical and basal media was removed and the amount of FITC-dextran quantified by measuring the absorbance at 492 nm using a FLUOstar Optima microtiter plate reader . Concentrations in apical and basal compartments were determined relative to the manufacturers’ standard curves.An amount of 1 μM AβCells were washed in 1 × HBSS, fixed in 4% PFA for 30 min at 4 °C and blocked in 5% NGS in 0.1% PBST for 1 h. Primary antibody was then applied at dilutions detailed in 4 onto 50 μg/mL fibronectin-coated Ibidi glass μ-slides. At ~80% confluence (3 days later), 1 μM Alexa Fluor-tagged Aβ1-42 or an equivalent volume of vehicle was applied for 3 h. For studies with LysoSensor Yellow/Blue DND 160, the slides were returned to the incubators for 24 h prior to application of the probe. For studies with Magic Red, the slides were returned to the incubators for 0.5, 3, 24 and 48 h prior to application of the probe. To evaluate lysosomal Aβ1-42 localization, 5 μM LysoSensor Yellow/Blue was applied for 5 min, the cells washed and imaged sequentially at ×63 magnification with a Leica SP8 confocal laser scanning microscope. Z-stacks were acquired across three random fields of view from three biological replicates. For analyzing the size of late-endocytic compartments, RPE cultures were labelled with Alexa Fluor-tagged Aβ1-42 and LysoSensor Yellow/Blue DND 160 as described before and the diameter of 25 vesicles was measured per field of view. A total of six images were acquired per condition across three biological replicates. To assess the effects of Aβ1-42 on lysosomal cathepsin B activity, Magic Red staining solution (x26) was applied at 0.5, 3, 24 or 48 h after Aβ exposure for 30 min at 37 °C with DMSO as a negative control. To assess the Magic Red response to POS, cultures were fed with isolated porcine POS at 4 μg/cm2 as described before [1-42 was quantified using Volocity software  which uses the Costes method [For live-cell imaging, cells were plated at 1 × 10d before ,29 for 35 cells/well in 6-well plates and maintained for two weeks to allow confluent cultures to develop. Total RNA was extracted using a Trizol method at 0.5, 3, 24 and 48 h following a 3 h exposure to oligomeric Aβ1-42. Briefly, cells were lysed in 500 μL TriIzol and subject to RNA/protein/DNA phase separation by the addition of 200 μL 1-bromo-3-chloropropane followed by centrifugation . RNA precipitation was by addition of 2 μL RNase-free glycogen and 250 μL isopropanol to RNA-containing supernatants for 20 min followed by centrifugation at 12,000× g for 10 min. Contaminants were removed by suspension in 500 μL 75% ethanol and centrifugation as before, after which RNA pellets were solubilized in RNAse free water. RNA yields were determined using a NanoDrop ND-1000 . RNA was transcribed into cDNA using the iScript cDNA synthesis kit  and qPCR was performed on a CFX96 Real-Time PCR detection system  using the SYBR green method. The 20 μL PCR reactions consisted of 0.6 μL 10 μM forward and reverse primers, respectively, 6.8 μL UltraPure™ DNase/RNase-Free distilled water , 10 μL iTaq Universal SYBR Green Supermix  and 2 μL of sample cDNA. Details of primers used are shown in −ΔΔCT methods. PCR products were resolved on a 1.2% agarose gel alongside a 100 bp DNA ladder at 120 V, 400 mA for 40 min to confirm primer specificity.ARPE-19 cells were seeded at a density of 1 × 102 1 mM MgCl2; pH 7.0) with 48% sucrose w/v, subject to agitation for 2 min and centrifuged at 5000× g for 5 min. The resulting supernatant was filtered through sterile surgical gauze into an Eppendorf tube containing an identical volume of KCl Buffer and solutions centrifuged at 4000× g for 7 min to isolate POS pellets. Covalent labelling with FITC was achieved by resuspension and 1 h dark rotation of isolated POS with 500 μL labelling buffer  and 150 μL FITC  per retina. FITC-POS pellets were obtained by centrifugation at 3000× g for 5 min and suspended in DMEM containing 2.5% sucrose w/v prior to use. Concentrations were determined by BCA assay  according to the manufacturer’s instructions.POS were isolated according to published procedures ,32,33. M4 in 50 μg/mL fibronectin-coated Ibidi glass bottom μ-slides 2 weeks prior to use in studies as described before [1-42 or an equal volume of vehicle was applied to post-confluent cultures for 3 h, washed and returned to the incubator for 23 h [2 for 2 h at 37 °C, 5% CO2 to facilitate maximum binding with minimal internalization [ARPE-19 cells were seeded at 1 × 10d before . An amoufor 23 h . FITC-POlization , after wlization ,36. Cultt-test or one-way ANOVA with Tukey’s multiple comparisons. Data where there was no indication of a Gaussian distribution were analyzed using the Mann–Whitney test or Kruskal–Wallis test with Dunn’s multiple comparison. Pairwise comparisons were two tailed. Data are expressed as the means ± with (n) representing the number of replicates (indicated in figure legends) with statistical significance denoted as * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 and *** = p ≤ 0.0001.Statistical analyses were performed using the GraphPad Prism Software . Normally distributed data were analyzed using either the Student’s 1-42. As Aβ levels in ocular fluids are reported to be in the picomolar to nanomolar range [1-42 in our experiments. A timeline of the experiment is shown in To study Aβ effects in living eyes, wild-type C57BL/6J mice were subretinally injected with a single dose of human oligomeric Aβar range , we usedWe also assessed the functionality of mouse retinas by longitudinal full-field scotopic ERGs. Electrophysiological readouts are routinely used as a measure of overall retinal activity and can provide evidence of functional deficits even in the absence of apparent morphological changes. Alterations associated with retinal aging and disease can therefore be detected by recording the amplitude/duration of A waves (derived from photoreceptor rods/cones) and B waves  after dark adaptation followed by a bright flash. ERG recordings were obtained with measurements set at the widest aperture to encompass the optic nerve and the site of the injection. Results show that within 2 weeks post-exposure, subretinal Aβ accumulation had no effect on global retinal function .In parallel, we tested for gross structural changes to mouse eyes using OCT. This non-invasive method provides detailed cross-sectional information on the thickness/structure of individual ocular layers across the scanned area. OCT revealed striking evidence of localized pathology in Aβ-treated eyes including subretinal fluid accumulation, areas of RPE hypertrophy and the presence of hyper-reflective material subretinally 1 week after injection i,k. Howe2 scan area, no significant differences were recorded between Aβ- vs. vehicle-injected eyes for any specific retinal layer or indeed for total retinal thickness when measured across the whole retina. Measurement of the lesion volume in each Aβ-injected eye showed a 0.52 ± 0.12 mm3 SEM area of damage at 1 week, and an area of 0.45 ± 0.16 mm3 SEM at 2 weeks  and disorganized inner segments (IS) b. DiscreMouse eyes were also probed for the low-density lipoprotein receptor-related protein 1 (LRP1) to assess whether Aβ had elicited a potential change in the expression of this well-known Aβ-clearance transporter a,b. Diff1-42, we recorded the percentage closure of the wound at the following time points: 6 h: 14.5 ± 5.1 SEM untreated, 23.4 ± 8.5 SEM vehicle and 17.5 ± 4.8 SEM Aβ-treated ; 24 h: 48.7 ± 5.2 SEM untreated, 58.1 ± 4.6 SEM vehicle and 74.4 ± 3.3 SEM Aβ-treated ; 48 h: 77.2 ± 3.0 SEM untreated, 85.3 ± 3.1 SEM vehicle and 94.1 ± 0.6 SEM Aβ-treated . One-way ANOVA with Tukey’s multiple comparisons test. Exposure to Aβ resulted in an increased rate of wound closure with significant differences recorded at 24 and 48 h compared to vehicle or untreated controls  using a scratch assay. After exposing CECs to either vehicle or 1 μM of human oligomeric Aβcontrols a,b.1-40 resulted in elevated Vascular Endothelial Growth Factor (VEGF) expression whilst reducing Pigment Epithelium-Derived Factor (PEDF) [1-42, and quantified the amounts of directionally secreted VEGF and PEDF in apical and basal Transwell compartments after 24 and 48 h  labelling, where foci of contracted cells resembling a lesion were particularly noticeable after 48 h following Aβ treatment  . We treaand 48 h . Surpris studies ,26. Give studies ,8, we quvitreous , and 119reatment . In ordey U test d–f. A ma1-42 after which LysoSensor was used to label late compartments in which trafficked cargos are most likely to be found [1-42 and its tagged Alexa Fluor probe, we also verified the strength of this conjugation using immunostaining. For this study, RPE cultures were exposed to 1 μM of fluorescently tagged human oligomeric Aβ1-42, after which presence of the conjugated molecules were independently verified using the 82E1 antibody. A 48 h time point following Aβ exposure was selected to reflect the longest experimental period within the intracellular environment  values for individual candidate reference genes were obtained for each time point and combined in a box and whisker plot to reflect the variability of mRNA expression across all time points/conditions. GAPDH: 32.9 ± 0.4 SEM, ACTB: 23.77 ± 0.2 SEM, CYC1: 27.14 ± 0.1 SEM and EIF4A2: 21.1 ± 0.1 SEM , mitochondrial cytochrome C1 (CYC1) , glycera 0.1 SEM . EIF4A2  0.1 SEM h, which 1-42 persisted within late compartments of RPE cells despite an upregulation of lysosomal cathepsin B.The Magic Red assay used to evaluate the lysosomal cathepsin B enzymatic response was also used to determine the dynamics of Aβ trafficking into late-endocytic compartments and to quantify  Aβ co-lo1-42, 24 h after which they were fed with POS, in keeping with the diurnal cycle  isoforms [1-42 effects appear to be mediated directly on CECs rather than through upregulation of VEGF by RPE cells. Our studies also demonstrate that exposure to physiological Aβ1-42 amounts led to contracted actin foci and elevated paracellular permeability in the RPE, consistent with an impaired blood–retinal barrier reported by others [Given the (1) well-established pro-angiogenic properties of Aβ, (2) the presence of choroidal Aβ staining observed in AMD tissues, as well as (3) Aβ effects in the choroid reported in other animal models , we sougtrations ,39. Expofeatures ,58,59,60ing PEDF . Interesisoforms ; supporty others ,54. Suchy others .1-42, as much as ~26% of the lysosomal signal was positive for Aβ after 24 h. The measured diameters of these late-endocytic compartments correlated with the reported size of lysosomes [Our work and that of others has previously shown that Aβ enters neurons and accumulates in lysosomes ,63. APP ysosomes . We founysosomes . Of noteysosomes ,68. Nextysosomes  importanysosomes ,71. A tiysosomes  does notysosomes ,66,72. Tysosomes ,40,73. Tysosomes , which wysosomes . A similysosomes . Our finysosomes , was med1-42, which persisted in lysosomes of neurons for several days to impair their activities [Next, we studied whether this lysosomal response successfully cleared Aβ cargos. Our findings showed that Aβ entry into lysosomes occurred over a prolonged period, with a maximal aggregation point at 24 h. However, clearance of Aβ cargos thereafter was slow, with only 15.9% of the maximal cargo cleared in the subsequent 24 h period. This revealed the following salient points. (1) The rapid and substantial burst of lysosomal cathepsin B activity was somewhat out of step with prolonged Aβ aggregation in these compartments. (2) Aβ clearance by cathepsin B, and presumably other lysosomal enzymes, removed only a small proportion of these cargos at least within assay timelines. 3) Prolonged Aβ aggregation in lysosomes and their slower clearance could result in the gradual build-up of Aβ within RPE cells over long periods. Indeed, the uptake of extracellular Aβ at low levels and their concentration as high molecular weight species in endosomes/lysosomes have been reported in neurons affected by Alzheimer’s disease . These ftivities . Althougtivities , deficie Prolongetivities . PreviouAMD is a multifactorial disease with an incompletely defined etiology. Currently, this common cause of blindness, which affects millions of individuals globally, has no effective treatment. The accumulation of the Alzheimer’s disease-associated Aβ proteins reported in donor aged and AMD eyes has prompted studies to elucidate its mode of action in the retina. Our findings show hitherto unreported AMD-like features in living mouse eyes as well as direct Aβ effects in endothelial cells. Studies in the RPE revealed evidence of lysosomal dysfunction alongside conditions favoring the intracellular accumulation of Aβ. These insights suggest impairment of cellular proteostasis including clearance mechanisms in the RPE, which is typically associated with the intracellular accumulation of pathogenic macromolecules. Our discoveries alongside findings from other groups indicate that targeting Aβ synthesis and clearance mechanisms in the retina may be a promising pathway towards developing a new treatment for AMD. Hence, re-purposing of anti-Aβ compounds developed for Alzheimer’s disease could be a useful approach.


To assess the role of sentinel lymph-node biopsy (SLNB) and FDG-PET in staging and radiation treatment (RT) of anal cancer patients. This retrospective study was performed on 80 patients  with a median age of 60 years (39–89 years) with anal squamous cell carcinoma who were treated from March 2008 to March 2018 at the IRCCS San Raffaele Hospital. Patients without clinical evidence of inguinal LNs metastases and/or with discordance between clinical evidence and imaging features were considered for SLNB. FDG-PET was performed in 69/80 patients. Patients with negative imaging in inguinal region and negative SLNB could avoid RT on groin to spare inguinal toxicity. CTV included GTV (primary tumour and positive LNs) and pelvic ± inguinal LNs. PTV1 and PTV2 corresponded to GTV and CTV, respectively, adding 0.5 cm. RT dose was 50.4 Gy/28 fractions to PTV2 and 64.8 Gy/36 fractions to PTV1, delivered with 3DCRT (n = 24) or IMRT (n = 56), concomitant to Mitomycin-C and 5-FU chemotherapy. FDG-PET showed inguinal uptake in 21/69 patients (30%) and was negative in 48/69 patients (70%). Lymphoscintigraphy was performed in 11/21 positive patients , and in 29/48 negative patients . Sensitivity, specificity, positive and negative predictive value of FDG-PET were 62%, 79%, 40% and 82%, respectively. Median follow-up time from diagnosis was 40.3 months (range: 4.6–136.4 months): 69 patients (86%) showed a complete response, 10 patients (13%) a partial response, 1 patient (1%) a stable disease. Patients treated on groin (n = 54) versus not treated (n = 26) showed more inguinal dermatitis . For patients treated on groin, G3–G4 inguinal dermatitis, stomatitis and neutropenia were significantly reduced with IMRT against 3DCRT techniques . SLNB improves the FDG-PET inguinal LNs staging in guiding the decision to treat inguinal nodes. IMRT technique significantly reduced G3-G4 toxicities when patients are treated on groin. Chemo-radiotherapy (CRT) is the standard treatment for anal SCC; whereas mesorectal and internal iliac lymph nodes (LNs) are routinely included in the radiation fields, the need of prophylactic inguinal irradiation is controversial.Squamous cell carcinoma (SCC) of the anal canal is a relatively uncommon tumour constituting only 4% of all lower alimentary tract and 2% of all digestive system malignancies. The tumour size (T stage) and the nodal status are recognized as the main prognostic factors2 and this is the main reason explaining the low sensitivity of either clinical examination in the detection of positive inguinal LNs.Accurate staging is crucial for radiotherapy (RT) treatment planning, aiming to select patients with localized disease. Fifteen-25% of patients with anal cancer show inguinal LNs metastases, while 44% of all LNs with positive metastases were reported to be smaller than 5 mm3. Two retrospective studies reported the inguinal recurrence rate in patients staged with conventional examinations who did not receive prophylactic inguinal irradiation: Blinde et al.4 showed inguinal recurrence rate of 0% in T1, 10% in T2 and 20% in T3–T4 patients with a median follow-up of 65 months, and Ortholan et al.5 reported an inguinal recurrence rate of 12% for T1–T2, and 30% for T3–T4 patients with a median follow-up of 61 months. As a consequence, the prescription of prophylactic inguinal irradiation based only on the T stage would cause an unnecessary irradiation of about 70–80% of T3–T4 patients, submitting them to a risk of severe acute toxicity, especially when using conformal RT techniques6. Conversely, about 10% of T1-T2 patients would be undertreated if inguinal irradiation is not prescribed. Thus, a better detection of initial inguinal LNs involvement would be important for selecting those patients who really deserve the inguinal irradiation. Recently, FDG-PET was shown to detect more abnormal inguinal LNs than clinical7, ultrasound inguinal8 or CT11 examination while sentinel lymph-node biopsy (SLNB) has been shown to be superior to both CT and FDG-PET in detecting metastatic LNs12.In a retrospective analysis of 270 patients who did not receive prophylactic inguinal irradiation, after a median follow-up of 72 months, the incidence of inguinal metastases was 8%5 but is unavoidably associated with increased toxicity. Better patient selection and the use of advanced RT techniques, such as intensity modulated RT (IMRT) and image guided RT (IGRT), may reduce toxicity13.Prophylactic RT including inguinal LNs irradiation was shown to decrease inguinal metastasis in several seriesIn the present study we reported our experience about the role of SLNB compared to FDG-PET in terms of disease staging and the clinical impact of inguinal irradiation with the use of advanced RT techniques.Eighty patients with histologically proven anal SCC treated between March 2008 and March 2018 were enrolled in a retrospective study. Patients’ ages ranged from 39 to 89 years with a median of 60 years. Of these 32 (40%) were male and 48 (60%) were female. Only 27/80 (34%) were HIV positive, and 21/27 (78%) were male. The main characteristics of patients are summarized in Table Patients underwent complete workup with full physical examination , proctoscopy or colonoscopy, chest-abdomen CT and pelvic MRI. Patients with T1N+, T2–T4 with any N, M0, Karnofsky performance status ≥ 70, adequate bone marrow, renal and hepatic function were candidate to CRT. TNM staging was T1 in 14, T2 in 30, T3 in 24 and T4 in 12 patients due to involvement of vagina (n = 8), prostate (n = 3) and uterus (n = 1). N staging was N0: 29, N1: 17, N2: 14, and N3:20 patients.Seven patients with limited metastatic disease were enrolled after collegial discussion and approval, despite the protocol violation , 2 patients: retroperitoneal LNs). The protocol was approved by our Institutional Ethical Committee , and all patients signed an informed consent. A retrospective comparison between FDG-PET inguinal LNs evaluation and SLNB results was made.Patients without clinical evidence of inguinal LNs involvement or with discordance between diagnostic imaging and clinical examination were considered for the SLNB protocol. The SLNB is a minimally invasive procedure which consists in submucosal injection of Technetium-99 labelled radio colloid around the primary tumour, patients in whom inguinal tracer uptake is detected undergo radio guided surgical removal of the inguinal sentinel LNs.15. Patients after positioning in supine position on Comby-Fix® underwent a contrast-enhanced CT and FDG-PET simulation. FDG-PET was used both for staging and target definition. CTV included the GTV (primary tumour and any positive LNs), the ischiorectal fossa, the mesorectum, and the internal and common iliac LNs until the L5-S1 space.The methods relative to lymphoscintigraphy, surgical removal and histopathological examination of sentinel LNs have been already described2 delivered from day 1 to 4 and from day 29 to 32 combined to Mitomycin-C 10 mg/m2 delivered on day 1 to 29.As the inguinal LNs irradiation is the standard treatment in anal cancer, all patients enrolled in this study with positive SLNB were treated on groin. Patients who had negative SLNB and negative imaging in inguinal region were considered for RT without groin irradiation, so as to spare inguinal toxicity; patients were also involved in the final clinical decision. PTV1 and PTV2 were defined as GTV and CTV, expanded with a margin of 0.5 cm, respectively. Median prescribed dose was 50.4 Gy in 28 fractions (1.8 Gy/fraction) to the PTV2, and 64.8 Gy in 36 fractions, delivered as sequential or concomitant boost, to the PTV1. RT techniques consisted of 3DCRT or IMRT (Volumetric Modulated Arc Therapy (VMAT) Rapid Arc or Helical Tomotherapy). The planned concomitant chemotherapy schedule was continuous infusion 5-FU 1,000 mg/mqSensitivity, specificity, area under the curve (AUC), positive and negative predictive values of FDG-PET against SLNB were assessed in the subgroup of patients that performed both examinations.Acute toxicities were scored according to the NCI-CTC for Adverse Events (Version 3); treatment interruptions were also reported. In order to assess the gain in avoiding to treat the inguinal LNs, a comparison in terms of inguinal LNs outcome and toxicity was made between the inguinal RT “IRT” and non-inguinal RT “NIRT” groups, also taking into account RT technique (3DCRT vs. IMRT).The differences between proportions  were tested by the two-tailed Fisher exact test.Time to progression (TTP) was calculated from the end of treatment and overall survival (OS) from the time of diagnosis to progression, last follow-up or death. Survival outcomes were evaluated using the Kaplan–Meier method. Statistical analyses were performed using MedCalc software .All procedures performed in studies involving human participants were in accordance with the ethical standards of the Institutional Research Committee and with the principles of the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.Sixty-nine/80 patients (86%) underwent FDG-PET that showed inguinal uptake in 21/69 patients (30%). Lymphoscintigraphy was performed in 11/21 (52%) patients: SLNB confirmed inguinal metastases in 4/10 patients (40%), 6/10 patients (60%) were false positive and SLN was not found in 1 patient. FDG-PET was negative in 48/69 patients (70%): lymphoscintigraphy was performed in 29/48 PET-negative patients: 5/28 (18%) showed metastasis despite the PET result, 23/28 (82%) were true negative and SLN was not found in 1 patient. The comparison between FDG-PET inguinal uptakes and SLNB is reported in Table The resulting sensitivity, specificity, positive and negative predictive value of FDG-PET were 62%, 79%, 40% and 82%, respectively as reported in Table All patients received the planned dose of 50.4 Gy in 28 fractions to the pelvis and ischiorectal fossa. The median dose of boost to T stage was 64.8 (61.4–72.4 Gy). Fifty-four/80 patients received prophylactic (n = 34) or curative (n = 20) inguinal irradiation (the “IRT” group) and 26/80 patients did not (the “NIRT” group). Boost was delivered sequentially in 96% of patients and concomitant in the remaining 4%. The median dose to the “IRT” group was 50.4 Gy (50.4–70.2 Gy). Twenty-five/26 patients of the “NIRT” group underwent SLNB procedure: 24/25 patients with histologically negative SLNB , and 1/25 SLN not found.In the “NIRT” group the percentage of patients with T1-T2 disease was higher than the percentage of patients with T3–T4 , while in the “IRT” group the rates were similar , as reported in Table For N staging we took into account any information from CT, MRI and FDG-PET. In the “NIRT” group the percentage of N0-N1 was higher than the percentage of N2–N3 , while in “IRT” they were similar 52% vs. 48%), as reported in Table 8%, as reMean duration of RT was 58 days (36–88 days): 42 patients (53%) needed a temporary suspension of RT (“IRT”: 29 patients and “NIRT”: 13 patients). Mean duration of suspension was 11 days (2–38 days); the mean duration in “IRT” was 11 days (2–38 days) and 12 days (3–23 days) in “NIRT”. Mean dose to suspension: 37.7 Gy (16.2–55.8 Gy) (“IRT”: 34.4 Gy (16.2–55.8 Gy) and “NIRT”: 44.7 Gy (34.2–57.6 Gy)).RT was delivered with 3DCRT in 24/80 patients (30%), 14/24 patients in the “IRT” group and 10/24 patients in the “NIRT” group. Fifty-six/80 (70%) patients were treated with IMRT: 18/80 patients (22%) were treated with Tomotherapy, ; 38/80 patients (47%) with VMAT .Concomitant Mitomycin-C and 5-FU was prescribed for 71/80 patients (89%), 2/80 patients (2.5%) received only Capecitabine and other 2/80 patients (2.5%) received Cisplatine. Median cycle of chemotherapy was 2 cycles (1 to 4 cycles). The 5/80 remaining patients (6%) who did not received concomitant chemotherapy: 3/5 patients were affected by hepatic cirrhosis and 2/5 patients were > 80 years old.There was no significant difference between G1–G2 and G3–G4 toxicities in “IRT” group compared to “NIRT” group except for inguinal dermatitis, as expected (G1–G2: 27/54 patients (50%) vs. 3/26 patients (12%), p = 0.001; and G3–G4: 9/54 patients (17%) vs. 0/26 patients (0%) (p = 0.03)). Toxicity data are reported in Table Sixty-nine/80 patients achieved the complete response, 10/80 patients had a partial remission and 1/80 patient had stable disease. Nineteen/80 patients had progression disease, 8/19 had local relapse , and 11/19 had only distant relapse. No patient treated or not on groin experienced inguinal relapse. Sixty-four/80 patients are alive and 16/80 patients died for progression disease at the last follow-up.Median time to local relapse was 38.9 months (4.8–123.2 months). Median time to progression was 35.2 months (3.0–133.0 months), excluded patients with metastasis at the time of diagnosis. Median OS from the time of diagnosis was 40.3 months (4.6–136.4 months), 46/80 patients (58%) achieved a 3 years OS and 32/80 patients (40%) were alive at 5 years from the end of treatment. Nine/80 patients underwent surgery , mean time to surgery was 12.1 months (3.1–23.3 months).5 it is clear that treating or not the inguinal LNs based only on the T stage has a large risk of over/under-treatment. A better detection of early inguinal LNs involvement could properly select patients who really deserve inguinal irradiation.CRT is the standard treatment for anal SCC. The indication of prophylactic inguinal irradiation is, however, controversial. According to the two largest retrospective studies7, inguinal ultrasound8, and CT11, and, more in general, changing the TMN stage in 41% of patients with anal cancer; thus it has been recommended for the initial staging16. The low incidence of metachronous metastases and the side effects after radiotherapy could not justify a prophylactic treatment. A refined staging system with precise identification of disease extent could allow individualized therapy, ensuring the accurate coverage of disease while sparing disease-free organs. Feasibility and efficacy of SLNB has been addressed by several reports and the clinical utility of this procedure in improving disease staging and selecting patients for inguinal radiation and changing the therapeutic plan has also been outlined15.FDG-PET detects more abnormal inguinal LNs than clinical examination1 compared FDG-PET and SLNB in 27 patients. Pathologic inguinal uptake was found in 7 patients. SLNB confirmed inguinal metastases in 3/7 (42%) vs. our 4/10 (40%) patients, with 4/7 (57%) false positives vs. 6/10 (60%) in our patients, and did not show inguinal metastases in the 20 patients with no pathologic inguinal uptake. They reported positive and negative predictive value equal to 43% and 100% respectively, quite consistent with the values found in current study  patients. Despite the relatively small number of patients of these studies and of our, results are quite consistent and show that: a) despite recommended for initial staging, the FDG-PET results on inguinal LNs should be interpreted with caution, showing a large number of false positives; b) SLNB can further improve inguinal staging reducing FDG-PET false positive and false negative rates. In the present study FDG-PET importantly showed a false positive rate of 60% (6/10 patients), and a false negative rate of 18% (5/28 patients) when compared to SLNB.Engledow et al.For these reasons, SLNB can be considered as a good tool when comparing new modalities such as FDG-PET.12 reviewed the literature and found 6 studies reporting SLNB positivity rate stratified by T stage: summing his results to the data from all these studies, SLNB found metastases in 6/27 (22%) T1 patients and in 17/98 (17%) T2 patients. In our population, selecting T1 and T2 patients without considering N stage, SLNB found metastasis in 2/14 (14%) and 6/30 (20%) patients, respectively. Considering patients with T1 and T2 N0 stage at diagnostic imaging, who would probably be selected to skip inguinal RT, SLNB showed metastases in 1/9 (11%) and 1/12 (8%) patients, respectively. In short, with the limitation of the small number of patients in the present study and in literature, SLNB seems to be the most effective procedure to select patients for inguinal RT.In particular, the reduction of the false negative rate could be very important, especially in case of stage T1-T2, as a positive SLNB would possibly suggest the radiation oncologist to treat the groins even in presence of a negative FDG-PET. Mistrangelo et al.3, the absence of inguinal relapse in the “NIRT” group indicates that SLNB is likely to be able to identify the true negative patients.Interestingly, with a median OS of 48.5 months (1.3–109.2 months) none of the 26/80 patients, who did not receive inguinal irradiation, had inguinal relapse. Considered that the median time of inguinal recurrence has been reported to be around 16 monthsAnother relevant issue concerns the impact of advanced RT techniques in sparing the normal tissues with/without treating the inguinal LNs. In the current study, the only significantly toxicity more frequent in the “IRT” group versus “NIRT” group was the inguinal dermatitis. Interestingly, the increase of inguinal dermatitis in the “IRT” group did not translate in an increased rate of treatment suspension, neither of the duration of the suspension compared to “NIRT” group. It is important to note that the median dose to suspension was 37.7 Gy, lower in “IRT” group than “NIRT” group . Genito-urinary and gastro-intestinal toxicities were similar in the two groups; in particular G3-G4 diarrhoea occurred in 3/54 and 2/26 patients, respectively.18, 23% and 11% in the RTOG 98116, respectively. Both trials used conventional RT techniques. The use of IMRT seems to reduce the incidence of G ≥ 3 GI toxicity in a range from 019 to 11–15.1%21, with most studies reporting an incidence from 7 to 9%23, without impairing the efficacy compared with traditional RT techniques24. A comparable range of G ≥ 3 GI toxicity, from 5 to 10%, was reported with the use of VMAT25 and Tomotherapy26. IMRT, VMAT and Tomotherapy, seem to reduce also G ≥ 3 genito-urinary toxicity to 0–3%27. Similarly to the two recent phase III trial, all the above reported trials using IMRT, VMAT or Tomotherapy, treated the inguinal LNs, then it is impossible to ascertain whether the exclusion of inguinal nodal area may translate into a further reduction of gastro-intestinal and genito-urinary toxicity.The incidence of G ≥ 3 diarrhoea and genito-urinary toxicity in the Mitomycin-C groups reported by the two more recent phase III trials of CRT for anal cancer was 9% and 2% in the ACT IISLNB staging of inguinal LNs seems to be superior compared to the FDG-PET, it allows to better identify false positive and false negative FDG-PET patients. Combination of FDG-PET and SLNB could guide the therapeutic decision and advanced RT techniques should better avoid toxicity, especially if patients are treated on groin.


The TFPrTMOS-derived membranes were deposited above a temperature of 175 °C, where the deposition behavior of TFPrTMOS was dependent on the organic functional groups decomposition temperature. The highest membrane rejection was from NaCl at 91.0% when deposited at 200 °C. For anions, the SO42− rejections were the greatest. It was also possible to separate monovalent and divalent anions, as the negatively charged groups on the membrane surfaces retained pore sizes >1.48 nm. Ions were also easily separated by molecular sieving below a pore size of 0.50 nm. For the TFPrTMOS-derived membrane deposited at 175 °C, glucose showed 67% rejection, which was higher than that achieved through the propyltrimethoxysilane membrane. We infer that charge exclusion might be due to the dissociation of hydroxyl groups resulting from decomposition of organic groups. Pore size and organic functional group decomposition were found to be important for ion permeation.The development of inorganic membranes has mainly found applicability in liquid separation technologies. However, only a few reports cite the permeation and separation of liquids through inorganic nanofiltration membranes compared with the more popular microfiltration membranes. Herein, we prepared silica membranes using 3,3,3-trifluoropropyltrimethoxysilane (TFPrTMOS) to investigate its liquid permeance performance using four different ion solutions (i.e., NaCl, Na This high rejection was said to be due to the molecular sieving effect.The technique of liquid separation using inorganic membranes has been one of the most compelling developments in recent decades, as they have the potential for highly selective separations through manipulation of their thermal and chemical properties ,2. In pa3+, Al3+, Mg2+, and SO42−, with hydration diameters of 0.91, 0.95, 0.87, and 0.76 nm, respectively. The ion rejections for Fe3+, Al3+, Mg2+, and SO42− were all found to be over 80% due to charge interactions between the ions and the surface of the zeolite membrane. Tsuru et al.  are the permeated molecules, t [s] is the permeation time, A [m2] is the membrane area, and Δp [Pa] is the pressure difference.The single gas permeation tests were carried out for gases H2SO4, MgCl2, and MgSO4 (nd MgSO4 . FurtherJ [kg m−2 h−1]) and the rejection (R [%]) were each calculated using Equations (2) and (3):pC and bC [mol L−1] are the concentrations of the permeate and feed solutions, respectively, and W [kg] is the total weight of the permeate solution.The solute concentrations were measured using a TOC-L total organic carbon meter  and an ES-71 electrical conductivity meter . The flux  of CF3–C2H4 decomposed around 120–230 °C. This equates to a total weight change percent of 22.8% and 10.1% around 120–230 °C and 230–300 °C, respectively. The molecular weight ratio of –CF3 and C2H4 was found to be 2.5, which is similar to a ratio of 22.8% and 10.1% (=2.3). Thus, it was concluded that CF3 decomposed around 120–230 °C and C2H4 decomposed around 230–300 °C.The TFPrTMOS powder decomposition did, however, show more sharp peaks compared with the decomposition of the PrTMOS powders in 2 permeances reduce compared with the γ-alumina substrate, indicating that the TFPrTMOS decomposed successfully to form the membranes. The highest H2/SF6 permeance ratio among the seven membranes studied was 1645 from a membrane deposited at 250 °C. For H2, N2, and SF6, the permeances were 4.01 × 10−7 mol m−2 s−1 Pa−1, 8.41 × 10−9 mol m−2 s−1 Pa−1, and 2.44 × 10−10 mol m−2 s−1 Pa−1, respectively. The H2 permeance for the membrane deposited at 270 °C was also found to be high at around 1.28 × 10−6 mol m−2 s−1 Pa−1, which was slightly lower than that of the γ-alumina substrate. The deposition temperature for the TFPrTMOS was between 175 °C and 270 °C.2 and SF6 over that of H2. Here, the pore size was found to differ only between 175 °C and 250 °C, as shown in 2H4 and CF3–C2H4 each decomposed at around 250 °C. The pores also hardly formed below 200 °C, with a temperature over 250 °C found to be needed to obtain larger pores. This trend was also discussed for the deposition of PrTMOS in other research work [rch work .−10 mol m−2 s−1 Pa−1 through the membrane deposited at 225 °C. The other water permeances through these membranes also showed similar trends, particularly for those of SF6, as shown in + and Cl− were determined to be 0.72 and 0.66 nm, respectively. The high rejections through the membranes in this case can be explained by molecular sieving. Nevertheless, the NaCl with a pore size of 1.48 nm had a rejection of 78.4% through the membrane deposited at 175 °C. Unfortunately, it is difficult to discuss this effect by molecular sieving only.As mentioned previously, the pore sizes of the membranes deposited between 200 °C and 250 °C were around 0.48–0.50 nm, whereas the hydrated diameters of Na2/SF6 permeance ratios. It is well known that glucose is a neutral molecule without charge effects in the presence of water. Here, we see the glucose rejection increase with an increase in the H2/SF6 permeance ratios up until a H2/SF6 ratio of 30, which indicates that both SF6 and glucose permeations are based on the molecular sieving mechanism. The maximum rejection was 84.6%. The molecular weight of glucose in this case was more than twice that of NaCl. Notwithstanding, the maximum NaCl rejection was 91.0%, which was higher than that of glucose. Thus, charge exclusion effects should be considered during permeations. This will be discussed using multivalent ions in the following section.2SO4, MgCl2, and MgSO4 were 91.0%, 95.8%, and 98.2%, respectively. Systematically, the order of these rejections was MgSO4 > MgCl2 > Na2SO4 > NaCl, as shown in 2+ rejections were the highest among the cations, whereas the SO42− rejections were found the highest among the anions. The rejections through the membrane deposited at 175 °C for NaCl, Na2SO4, MgCl2, and MgSO4 were 78.0%, 96.2%, 70.1%, and 93.0%, respectively. The rejection order was Na2SO4 > MgSO4 > NaCl > MgCl2. The average pore size was also found to be 1.48 nm, which is larger than that of the membrane deposited at 250 °C. Negatively charged polymer commercial nanofiltration (molecular weight cutoff: 1000–2000) also showed the same ordering at Na2SO4 > MgSO4 > NaCl > MgCl2, which is the same order as the membrane deposited at 175 °C. Through the negatively charged membranes, SO42− rejections were the highest among the anions, whereas the Mg2+ rejections were found the lowest among the cations [4 and MgCl2 that included the ion with the largest hydrated diameter . The rejections for the membrane deposited at 270 °C for NaCl, Na2SO4, MgCl2, and MgSO4 were 56.7%, 79.3%, 21.6%, and 44.6%, respectively. The order of this rejection was Na2SO4 > NaCl > MgSO4 > MgCl2. The pore size found for the membrane deposited at 270 °C was also the largest among the three membranes, as shown in 2+ rejections were lower than the Na+ rejections. cations . In addi2SO4, MgCl2, and MgSO4 rejections of the PrTMOS-derived membranes were 92.0%, 97.4%, 69.2%, and 85.4%, respectively. The glucose rejection was only 35.9%, whereas that of the TFPrTMOS-derived membrane was 67.2%. The order of the rejection was found to be Na2SO4 > NaCl > MgSO4 > MgCl2, which was different from that of the TFPrTMOS-derived membrane, as shown previously. The order was also the same as the membrane deposited at 270 °C. 2 permeance of the PrTMOS-derived membrane was found to be smaller than that of the TFPrTMOS-derived membrane. However, the permeation ratios of H2/N2 and H2/SF6 were shown to be 3.6 and 5.5, indicating that the permeation may be based on Knudsen diffusion. This may explain why the pore size was similar to that of the TFPrTMOS-derived membrane deposited at 270 °C. At the same deposition temperature, TFPrTMOS was found to generate much smaller pores than PrTMOS. This means that TFPrTMOS deposits more easily than PrTMOS because of its higher reactivity. The decomposition temperature of C3H7 within PrTMOS was 100–200 °C, as shown in 3H7 should also decompose at 175 °C to form hydroxyl groups inside the separation layer. The permeation properties of the PrTMOS-derived membrane were also similar to those of the TFPrTMOS-derived membrane deposited at 270 °C. We found the pore size and organic group decomposition to be the most important factors for ion permeation.Both PrTMOS- and TFPrTMOS-derived membranes deposited at 175 °C were compared through ion permeation. The NaCl, Na2/SF6 permeance ratio (H2/SF6 = 1645) was found to be at 250 °C. Here, NaCl rejections were shown to improve around 175–250 °C. Ion rejections were controlled by the decomposition temperature due to the decomposition of the organic groups in the silica precursors. The NaCl had a rejection of 91.0%, which was obtained through the membrane deposited at 200 °C. We infer the TFPrTMOS membranes to be negatively charged. The ion permeation mechanism was found to change due to charge effects with membranes having a pore size of 1.48 nm or above. The TFPrTMOS membrane showed higher glucose rejection at 67.2%, in comparison with the PrTMOS membrane. The pore size and the decomposition of the organic groups were found to be important for ion permeation.In this study, TFPrTMOS membranes were deposited successfully at a temperature higher than 175 °C. The TFPrTMOS membrane that had the highest H


We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced  Summary: We have designed an optimized and integrating inducible-promoter system to control the timing, spatial domains, and levels of gene misexpression throughout avian development. Also, we show that transgene expression levels can be modulated by titrating the concentration of dox, and precise spatial control over transgene activation can be achieved by implanting beads or gels that release dox locally. Thus, our optimized and integrating inducible-promoter system can control the timing, spatial domains, and levels of gene misexpression throughout avian development, which will be useful for a broad range of experimental contexts.Building on the clear advantages of inducible promoter systems for exerting spatiotemporal control over gene expression and the ability of transposable elements to integrate into the avian genome and facilitate long-term expression throughout development , we endet genome . We furtt genome  and a DNt genome . We findTo maximize transfection and electroporation efficiency we aimed to generate plasmids as small as possible. Smaller plasmids have been shown to transfect and electroporate more efficiently than large plasmids . MoreoveWe chose the PGK1 promoter over other commonly used promoters due to its relatively small size at 500 bp and its consistent expression across different cell types . Moreovein ovo development (e.g. 21 days for chick and 28 days for duck). To ensure stable and robust expression over the course of embryogenesis, we used a type II transposon (cut and paste) system to integrate sequences into the genome  . The ITRs in the presence of piggyBac transposase (PBase) semi-randomly integrates into the host genome at sequences containing a TTAA motif through a cut and paste mechanism. We used the enhanced piggyBac sequence which contains two point mutations in the left ‘5 ITR that increase transposition efficiency  B. This cClover3) . As a maClover3)  to expreClover3) . An optiClover3)  can be aficiency . To exprficiency C. This pficiency  under thRunx2, 1419 bp), an extracellular matrix molecule , and a cytokine , into pEPIC1.1. We first confirmed that pEPIC1.1 constructs could over-express our genes of interest by transfecting them into a chick fibroblast cell line (DF-1). We found that pEPIC1.1-Runx2, pEPIC1.1-Mmp13, and pEPIC1.1-Cxcl14 all induce strong over-expression compared to empty pEPIC1.1 (Runx2 construct increased Runx2 mRNA levels 27±4.3 times by qPCR (P<0.005) and RUNX2 protein levels 23±2.7 times by WB compared to pEPIC1.1 (P<0.005) (Mmp13 construct increased Mmp13 mRNA levels 480±2.4 times by qPCR (P<0.005) and the MMP13 protein levels 5±0.96 times by WB compared to pEPIC1.1 (P<0.005)  and the CXCL14 protein levels 32±4.6 times by WB compared to pEPIC1.1 (P<0.005)  D. The pEP<0.005) E. The pEP<0.005) F.P<0.05)  G,H. By dCxcl14 with or without hyPBase, and then electroporated. Mandibles were then cultured over 7 days. After 5 days of culture, mandibles electroporated with pNano-hyPBase retained strong GFP expression while mandibles without pNano-hyPBase had greatly reduced expression compared to 1-day post-electroporation  were dissected from HH24 chick embryos, injected with a plasmid solution containing pEPIC1.1-poration I. After piggyBac vectors we generated a general piggyBac cloning vector pPID2   A. pPID2 utations , a DTS, 4–106 molecules of plasmid DNA per cell)  . To overer cell) , 2005. Aer cell)  because er cell) , is smaler cell) , and doeer cell) . The DTSer cell) .piggyBac transgene expression  construct as a minimal dox-inducible plasmid  transactivator (rtTA) and mNeonGreen (GFP) under the PGK promoter , Runx2, and Mmp13. We first transfected DF-1 cells with pPIDNB-Cxcl14, treated with various doses of dox, and found that Cxcl14 expression correlated with the concentration of dox (Cxcl14 mRNA expression by 27±6.4 (P<0.05), 96±23 (P<0.05), 149±34 (P<0.05), and 178±20 (P<0.005) times, respectively, compared to cells not treated with dox (P<0.005), 12±3.8 (P<0.05), 15±1.6 (P<0.005), and 17±1.9 (P<0.005) times, respectively, compared to cells not treated with dox (We next tested the ability of pPIDNB to drive exogenous gene expression by cloning in the coding sequences for n of dox F. We fouwith dox F. WB anawith dox G. These with dox .Gas1 and pNano-hyPBase. DF-1 cells were passaged over 4 weeks and then fluorescence-activated cell sorted (FACS) for GFP to confirm pPIDNB-Gas1 could be stably integrated into the host genome and remain dox-inducible. We treated cells with dox and found that they were induced in a dose-response manner. After treating cells with 1, 5, 10, and 50 ng/ml dox for 24 h, Gas1 mRNA expression increased by 1.1±0.16 (P>0.05), 23±0.99 (P<0.005), 34±2.7 (P<0.005), and 97±7.6 times (P<0.005), respectively, compared to cells not treated with dox  times more Runx2 mRNA and 30±3.2 (P<0.005) times more Mmp13 mRNA than cells transfected with empty pPIDNB, respectively (Runx2 and pPIDNB-Mmp13 expressing 4.4±1.1 (P<0.05) and 1.9±0.25 (P<0.05) times more RUNX2 and MMP13 protein than pPIDNB alone, respectively.To determine if pPIDNB can stably integrate into the genome and express a transgene, we transfected DF-1 cells with pPIDNB-with dox H. To conectively I,J. WB ain ovo, so that we can minimize the number of times we handle samples.Even though we found that 10 ng/ml of dox provides for high levels of induction, in order to achieve prolonged and robust gene expression in our subsequent long-term experiments, we decided to use 50 ng/ml dox. This higher concentration takes into account the half-life of dox, which is between 24–48 h in culture (based on estimates from the manufacturer), and our need to maintain gene expression for extended periods of time (like up to 10 days) without having to re-introduce additional dox, especially Gas1 or pPIDNB2-Gas1. Cells were passaged for 4 weeks and then sorted for GFP to generate stable lines with either pPIDNB-Gas1 or pPIDNB2-Gas1 integrated into their genomes. Cells with integrated pPIDNB-Gas1 or pPIDNB2-Gas1 were visualized by GFP. pPIDNB-Gas1 cells showed GFP localized throughout the entire cell while pPIDNB2-Gas1 showed nuclear localization of GFP  analog, and induce the tet expression system  cell cycle sensor sequence  to the din ovo in migrating NCM destined for the mandibular primordia (Movie 1).To exert spatiotemporal control over gene expression in embryonic tissues, we unilaterally electroporated the presumptive cephalic NCM of HH8.5 chick embryos with pPIDNB and pNano-hyPBase. At HH10, we assayed for the extent of electroporation by visualizing GFP-positive cells rimordia A. These -Gas1 and pNano-hyPBase and were treated with 50 ng/ml dox in ovo at HH15. By HH24, we observed RFP expression throughout the mandibular primordia . These embryos were then allowed to develop for 9 more days (to around HH40), at which point we observed robust unilateral RFP expression in the lower jaw . Pluronic F-127 is a liquid at low temperatures (4°C) but solidifies at higher temperatures (37°C) and has been used for delivering drugs to different tissues . The 3× FLAG 2× Strep tag sequence was amplified from AAVS1 Puro Tet3G 3× FLAG Twin Strep  . The mScarlet-I sequence was amplified from pmScarlet-i_C1   were amplified using Q5 Hot Start High-Fidelity DNA Polymerase . Fragments were cloned together using NEBuilder HiFi DNA Assembly Master Mix . EcoRI, XhoI, and EcorV restriction enzyme sites were incorporated as tails added to the primers. To generate pEPIC1.1, the enhanced , 92099) . mClover, 92099) . To gene, 85044) . To gene, 85044)  using KORunx2, Mmp13, and Cxcl14, RNA was extracted from DF-1 cells and HH27 whole chick heads using the RNeasy Plus Kit  following the manufacturer's directions. Whole heads and DF-1 cells were resuspended in 600 μl of RTL plus buffer supplemented with 1% β-mercaptoethanol. Homogenization was carried out in a Bead Mill 24  at 5 m/s for 30 s. Following purification of total RNA, residual genomic DNA was removed using TURBO DNA-free Kit . For RNA extractions involving Gas1, the PicoPure RNA Isolation Kit  was used following the manufacturer's directions and homogenization was carried out in a Bead Mill 24  at 4 m/s for 15 s.For Runx2, Mmp13, Cxcl14, and Gas1 were amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase  and cloned using CloneJET PCR Cloning Kit . Following confirmation of cloning of full length coding sequences by Sanger sequencing, Runx2, Mmp13, Cxcl14, and Gas1 were cloned into pEPIC1.1 digested with AflII  and EcoRI  or pPIDNB digested with AflII  and PstI  using NEBuilder HiFi DNA Assembly Master Mix. All constructs were verified by Sanger sequencing and midipreped for electroporation and transfection using PureLink Fast Low-Endotoxin Midi Kit .Full length cDNA synthesis from RNA was carried out using Maxima H-reverse transcriptase  following the manufacturer's directions with 2 μg of total RNA and 100 pmol of d(T)20 VN primer. The cDNA synthesis reaction was carried out at 50°C for 30 min, 55°C for 10 min, 60°C for 10 min, 65°C for 10 min, and 85°C for 5 min. Full length Gallus gallus) and duck (Anas platyrhynchos) were purchased from AA Lab Eggs  and incubated at 37.5°C in a humidified chamber  until they reached embryonic stages appropriate for manipulation and/or analyses. For all experiments, we adhered to accepted practices for the humane treatment of avian embryos as described in S3.4.4 of the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition  according to the manufacturer's instructions. Transfections for integrating piggyBac vectors were carried out in six-well plates with 5 µg piggyBac plasmid, 5 µg of pNano-hyPBase, and 20 µl of P3000.Embryonic chick fibroblasts (DF-1) were purchased  and cultured in Dulbecco's Modified Eagle's Medium  supplemented with 10% FBS  and 1× penicillin-streptomycin  at 37°C with 5% COElectroporations were performed by injecting a solution of pEPIC1.1-Cxcl14 and pNano-hyPBase at 3 µg/µl and 1 µg/µl, respectively, with a small amount of Fast Green dye. DNA was injected with a Pneumatic PicoPump  into dissected HH21 mandibular primordia using thin wall borosilicate glass micropipettes  pulled on a micropipette puller . Mandibles were placed between two gold plate electrodes 0.5 cm apart submerged in Hanks' balanced salt solution . Electroporations were carried out by delivering five square pulses at 25 V for 50 ms spaced 500 ms apart . Mandibles were then cultured in BgJB medium  supplemented with 10% FBS  and 1× penicillin-streptomycin .In ovo electroporations were performed using a solution of pPIDNB and pNano-hyPBase at 3 µg/µl and 1 µg/µl, respectively. With the addition of Fast Green tracer dye, DNA solution was injected into HH8.5 chick neural tubes with a Pneumatic PicoPump using thin wall borosilicate glass micropipettes pulled on a micropipette puller. Platinum electrodes were positioned on each side of the area pellucida, centered along the neural folds of the midbrain-hindbrain boundary as done previously to target the presumptive NCM destined for the mandibular arch . Gene expression was quantified by qPCR with iQ SYBR Green Supermix  and normalized to 18S rRNA following previously published protocols . Primer DF-1 cells were lysed with 1× RIPA lysis buffer  containing Halt protease inhibitors . A BCA assay  using a SpectraMax M5 plate reader  was performed to quantify protein, and 40 µg protein was electrophoresed on a 10% SDS polyacrylamide gel following a published protocol . Proteinin ovo treatments, 2.5 µl (for chick) and 3.75 µl (for duck) of the 1 mg/ml dox stock solution was diluted with 750 µl of HBSS. This solution was then gently pipetted into the egg adjacent to the embryo and allowed to diffuse.Stock solutions of doxycycline hyclate  were made to a final concentration of 1 mg/ml in water, filter sterilized, and stored at −20°C as single use aliquots. DF-1 cells and mandibles were treated in culture with the stock solution diluted in DMEM, with minocycline microspheres (Arrestin) added directly to each well, or by suspending microspheres in PBS and injecting them into the lower jaw with a 30-gauge needle. Pluronic F-127  was dissolved at a final concentration of 35% (w/v) in DMEM growth medium rocking at 4°C for 48 h. Dox was added to Pluronic F-127 for a final concentration of 500 ng/ml and injected into the lower jaw with an 18-gauge needle. For 2. All DF-1 experiments were carried out in six-well plates  with 2 ml of DMEM. Lower jaw time-lapse experiments were carried out on six-well transwell membranes  with 2 ml of DMEM. Brightfield and fluorescent images of duck HH24 mandibular primordia were captured on an epifluorescent stereomicroscope .DF-1 cells were imaged using a macroconfocal . Time-lapse experiments were carried out in a custom-made stage top incubator  set to 37°C, 95% humidity and 5% CODF-1 cells were washed with 2 ml of Trypsin followed by 3mL fresh wash. Trypsin activity was inhibited by adding 5 ml of DMEM with 10% FBS. Cells were pipetted and passed through 70 µm filter. Cells were sorted on FACSAriaII Flow Cytometer . For all sorts, debris and dead cells were eliminated using FSC-A and SSC-A gating, doublets were excluded via gating discrimination using FSC-H and FSC-W, and only GFP+ cells were collected.t-test was performed . When multiple comparisons were made, P-values were adjusted using the Holm–Bonferroni method (Statistical analysis carried out using Student's i method . We aime


G6PD mutations in the Lao Theung population were performed in this study.The prevalence and genotypes of G6PD deficiency vary worldwide, with higher prevalence in malaria endemic areas. The first-time assessment of G6PD deficiency prevalence and molecular characterization of G6PD gene mutations. The amplification refractory mutation system (ARMS)-PCR for detecting G6PD Aures was developed.A total of 252 unrelated Lao Theung participants residing in the Lao People’s Democratic Republic (PDR) were recruited. All participant samples were tested for G6PD enzyme activity and G6PD mutations were detected in 11.51% (29/252) of the participants. Eight G6PD mutations were detected. The G6PD Aures was the most common mutation identified in this cohort, which represented 58.62% (17/29) of all mutation. The mutation pattern was homogenous, predominantly involving the G6PD Aures mutation (6.75%), followed by 1.19% G6PD Union and 0.79% each G6PD Jammu, G6PD Mahidol and G6PD Kaiping. One subject (0.4%) each carried G6PD Viangchan and G6PD Canton. Interestingly, one case of coinheritance of G6PD Aures and Quing Yan was detected in this cohort. Based on levels of G6PD enzyme activity, the prevalence of G6PD deficiency in the Lao Theung population was 9.13% (23/252). The prevalence of G6PD deficient males and females (activity < 30%) in the Lao Theung population was 6.41% (5/78) and 1.72% (3/174), respectively, and the prevalence of G6PD intermediate (activity 30–70%) was 5.95% (15/252).The The G6PD Aures mutation is highly prevalent in the Lao Theung ethnic group. The common G6PD variants in continental Southeast Asian populations, G6PD Viangchan, Canton, Kaiping, Union and Mahidol, were not prevalent in this ethnic group. The technical simplicity of the developed ARMS-PCR will facilitate the final diagnosis of the G6PD Aures. X chromosome and is composed of 13 exons and 12 introns [G6PD gene, resulting in reduced enzyme activity and stability [Glucose 6˗phosphate dehydrogenase (G6PD) deficiency is the most common hereditary enzymopathy affecting people worldwide. G6PD is a key enzyme in the pentose phosphate pathway (PPP), a metabolic pathway that produces nicotinamide adenine dinucleotide phosphate (NADPH) to maintain the level of a reduced glutathione, which is important for defending against oxidative damage in red blood cells , 2. The tability , 5. G6PDPlasmodium vivax and Plasmodium ovale [Most people with G6PD deficiency do not exhibit symptoms unless exposed to an oxidation-inducing agent or an infection . G6PD deum ovale . Beyond um ovale , 12.G6PD mutations have been reported [G6PD gene, particularly in Asia, Africa, the Middle East and the Mediterranean [G6PD genotypes show population specificity. For example, in Southeast Asia, the G6PD Mahidol mutation is predominant among the Myanmar and Mon populations [G6PD mutations in the Lao Theung ethnic group is an important public health issue.More than 215 reported . The prereported , 14. Curerranean , 15, 16.ulations , while tulations , Cambodiulations  and Lao ulations –25. No sulations . The Laoulations . In addiulations . This stPeripheral blood samples were collected from 252 unrelated healthy Lao Mon-Khmer people, including 174 females and 78 males. Participants’ ages ranged from 18 to 50 years old. The participants were living in the Feuang District, Vientiane Province, and Lao PDR. This study was a community-based survey which was approved by the Ethics Committee of the university of Health Sciences, Lao PDR. The sample collection was performed in December 2016. Written informed consent was obtained before blood sampling from all individual participants included in the study.The blood samples were stored at 4 ºC and analysed for G6PD enzyme activity within 7 days after collection. All samples were measured for G6PD enzyme activity in duplicate using a quantitative G6PD kit . The assay was carried out at 30 ºC, and the method was performed following the manufacturer’s instructions. The haemoglobin (Hb) concentration was determined using a HemoCue Haemoglobin System . The G6PD enzyme activity values were calculated in units per gram of Hb (U/g Hb).G6PD mutations detected in Asia using a multiplex allele-specific PCR-based assay (DiaplexC™ G6PD Genotyping Kit (Asian type), SolGent, Daejeon, Korea). The genomic DNA of all blood samples was extracted by using a QIAamp® DNA Blood Kit  according to the manufacturer’s recommended protocol and kept at -80 °C until use. The commercial kit enabled the detection of 8 common G6PD mutations, including Vanua Lava c.383 T > C, Mahidol c.487 G > A, Mediterranean c.563 C > T, Coimbra c.592 C > T, Viangchan c.871 G > A, Union c.1360 C > T, Canton c.1376 G > T and Kaiping c.1388 G > A. The PCR mixture contained 12 µl of 2X Multiplex PCR Smart Mix, 2 µl of primer mixture, 20–50 ng DNA template and nuclease- free water to obtain a total volume of 25 µl. The PCR cycling conditions used were as follows: initial denaturation at 95 °C for 15 min, followed by 30 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 sec and extension at 72 °C for 40 sec; this was followed by a final extension at 72 °C for 5 min. After PCR amplification, 5 µl of the PCR product and 5 µl of a standard marker were separated by 3% agarose gel electrophoresis, stained with SYBR® Green  and visualized under UV light. The positive results were validated by PCR sequencing.All 252 subjects were genotyped for the 8 common G6PD gene mutation in the G6PD-deficient samples that could not be identified by DiaplexC™ G6PD Genotyping Kit. The entire coding sequence (exons 2–13) of the G6PD gene was amplified with specific primers following a protocol previously described [Bioneer, Daejeon, Korea) and subsequently sequenced by using the Sanger sequencing method . The sequence of G6PD was analysed by BLAST and compared to the G6PD gene mutation with database accession no. NC_000023.11.PCR sequencing was performed to identify the escribed . The PCRThe ARMS-PCR method for detecting the G6PD Aures mutation was first developed in this study. The G6PD Aures allele-specific PCR primer pair consisted of a forward primer (5′-ACCTGGCCAAGAAGAAGAT-3′) and a reverse primer (5′-CTCACTCTGTTTGCGGATG-3′), which produce a 226 bp fragment. Two additional primers consisting of a forward primer (5′-TGGTTCTGCCCTCTCTAC-3′) and a reverse primer (5′-GAGACACGGACAGA CAGA-3′) were added to produce a 519 bp fragment of the 3′-UTR region to serve as an internal amplification control  by using IBM SPSS statistics software version 22 . The 95% confidential intervals were calculated by Java Stat Binomial Confidence Intervals. The adjusted male median G6PD activity was calculated to identify the cut-off values for G6PD deficiency following a calculation method described previously . In detaG6PD gene mutations in this Lao Theung group was 11.51% (29/252), equating to 6.41% (5/78) in males and 13.79% (24/174) in females. Eight G6PD mutation sites were detected , followed by 1.19% G6PD Union c.1360C > T and 0.79% each G6PD Jammu c.871G > A with nt1311C, G6PD Mahidol c.487G > A and G6PD Kaiping c.1388G > A. One subject (0.4%) each carried G6PD Viangchan c.871G > A and G6PD Canton c.1376G > T . Fifteen female subjects with G6PD intermediate activity consisted 10 heterozygous mutations and 5 subjects cannot identify G6PD mutation. Eleven subjects carried heterozygous of the G6PD mutation had normal G6PD enzyme activity , ranging from 0.01 to 25.73 U/ gHb. The median and IQR of the G6PD activity of males and females were 6.68 U/gHb; IQR: 5.85–8.03 U/gHb and 7.21 U/gHb; IQR:6.22–8.34 U/gHb, respectively. The cut-off value for G6PD deficiency (activity below 30% of the AMM) in this cohort was < 2.0 U/gHb . Knowledge of the prevalence of G6PD variants among different ethnic groups is limited. The present study is the first to report the prevalence of G6PD deficiency and G6PD mutations in the Mon-Khmer or Lao Theung ethnic group. The prevalence of G6PD deficiency reported in this study (9.13%) is higher than that previously reported for unspecified ethnic Lao populations  [nd 7.2%) , 23, 25 G6PD mutation screening, the study of G6PD mutations focused only on the detection of the G6PD Viangchan mutation or identified the G6PD mutation only in males that presented with severe haemolytic anaemia [G6PD mutation have sufficient enzyme activity, they can pass an X-linked G6PD mutation to all of their sons and daughters along with a risk of developing the symptoms associated with a severe G6PD deficiency.However, the prevalence of G6PD deficiency in Lao populations was lower than that reported in neighboring Southeast Asian populations, including Mon 12%) , 30, Kar anaemia , 21, 23.% , 30, K anaemia –23, 25 aG6PD gene, eight G6PD mutations were detected in this Lao Theung group. A comparison of the G6PD mutation data collected in this and previous studies of non-specified Lao ethnic populations is summarized in Table G6PD mutation in the Lao Theung population was G6PD Aures c.143 T > C (6.75%); this result is different from that in a previous report, which found that the G6PD Viangchan mutation was the most common mutation in the Laotian population (1.21–6.76%) [G6PD mutation was not identified in five G6PD-deficient participants. The mutation site of those 5 cases may exist in the intron region, which could not be analyzed by our method.Regarding the mutation characteristic of the 1–6.76%) –25. The lation 1.–6.76% [2lation 1.–6.76% [21–6.76%)  and the 1–6.76%) . These d1–6.76%) , while G1–6.76%) , Cambodi1–6.76%)  and Viet1–6.76%) , but not1–6.76%) . The G6PThe G6PD Aures mutation is commonly detected in Mediterranean populations, such as the Saudi Arabian (11.2–20%) –36, UnitHowever, severe G6PD deficiency was observed for the first time for the Aures mutation in this study. The hemizygous males and homozygous females for the G6PD Aures mutation presented with a severe G6PD deficiency, whereas the phenotype of G6PD Aures heterozygous females varied from normal to moderately G6PD deficient. The G6PD Aures is located between the sites of G6PD Vietnam-1 and Vietnam-2/Bahia, near G6PD Rignano, which is distant from the protein domain for NADP-1 binding . The mutThe G6PD Aures mutations were detected in both male and female participants. As the inheritance of G6PD is X-linked, it is very important to address the finding that both homozygous and heterozygous G6PD females can pass the abnormal gene to their male child, leading to the risk of clinically presenting the G6PD Aures mutation. The knowledge about the effect of the G6PD Aures mutation is now still limited, and it is very important to test the haemolytic risk of anti-malarial drugs and other oxidative damage related to the G6PD Aures mutation in further studies as the Lao Theung people stay around the forest areas and have a risk of malaria infections.G6PD gene flanking the G6PD Aures mutation site must be amplified by PCR, purified and sent for sequencing analysis. Compared to the PCR sequencing method, the ARMS-PCR developed in this study can show a positive or negative G6PD Aures result in a shorter time with lower costs; however, this method cannot be used to determine the zygocity of the G6PD Aures. By using the ARMS-PCR method developed in this study, the G6PD Aures mutation was additionally detected in 7 female participants. This new method should be applicable in routine clinical practice for the detection of the G6PD Aures.This study reports the development of ARMS-PCR, which can detect the G6PD Aures in one PCR-electrophoresis step. The diagnosis of G6PD Aures could be alternatively performed by PCR sequencing analysis, as the G6PD mutation pattern identified in the Mon-Khmer group is quite homogenous and unique, predominantly involving the G6PD Aures mutation. This finding shows a founder effect in this population. The hemizygous males or homozygous females for the G6PD Aures mutation presented with a significant severe G6PD deficiency. Malaria infection, dengue fever and typhoid fever are prevalent in Lao PDR and knowing the G6PD status in local health areas, especially Lao Theung group, helps improve the management of acute haemolysis related to these infections.The