Escherichia coli (UPEC) proliferate within superficial bladder umbrella cells to form intracellular bacterial communities (IBCs) during early stages of urinary tract infections. However, the dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. We develop a human bladder-chip model wherein umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively, and bladder filling and voiding mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel to sites of infection leads to swarm and neutrophil extracellular trap formation but does not prevent IBC formation. Subsequently, we tracked bacterial growth dynamics in individual IBCs through two cycles of antibiotic administration interspersed with recovery periods which revealed that the elimination of bacteria within IBCs by the antibiotic was delayed, and in some instances, did not occur at all. During the recovery period, rapid proliferation in a significant fraction of IBCs reseeded new foci of infection through bacterial shedding and host cell exfoliation. These insights reinforce a dynamic role for IBCs as harbors of bacterial persistence, with significant consequences for non-compliance with antibiotic regimens.Uropathogenic  Escherichia coli , which either float freely in the urine and wash away when the bladder empties, or form communities inside cells that the bladder struggles to clear. It is possible that the bacteria living within cells are also more protected from the immune system and antibiotics. But this is hard to study in animal models.Urinary tract infections are one of the most common reasons people need antibiotics. These bacterial infections are typically caused by uropathogenic To overcome this, Sharma et al. built a ‘bladder-chip’ which mimics the interface between the blood vessels and the tissue layers of the human bladder. Similar chip devices have also been made for other organs. However, until now, no such model had been developed for the bladder.On the chip created by Sharma et al. is a layer of bladder cells which sit at the bottom of a channel filled with diluted human urine. These cells were infected with UPEC, and then imaged over time to see how the bacteria moved, interacted with the bladder cells, and aggregated together. Immune cells from human blood were then added to a vascular channel underneath the bladder tissue, which is coated with endothelial cells that normally line blood vessels. The immune cells rapidly crossed the endothelial barrier and entered the bladder tissue, and swarmed around sites of infection. In some instances, they released the contents of their cells to form net-like traps to catch the bacteria. But these traps failed to remove the bacteria living inside bladder cells.Antibiotics were then added to the urine flowing over the bladder cells as well as the vascular channel, similar to how drugs would be delivered in live human tissue. Sharma et al. discovered that the antibiotics killed bacteria residing in bladder cells slower than bacteria floating freely in the urine. Furthermore, they found that bacteria living in tightly packed communities within bladder cells were more likely to survive treatment and go on to re-infect other parts of the tissue.Antibiotic resistance is a pressing global challenge, and recurrent urinary tract infections are a significant contributor. The bladder-chip presented here could further our understanding of how these bacterial infections develop in vivo and how good antibiotics are at removing them. This could help researchers identify the best dosing and treatment strategies, as well as provide a platform for rapidly testing new antibiotic drugs and other therapies. Escherichia coli (UPEC), the causative agent for the majority of UTIs, exhibits a complex lifestyle in the bladder, with planktonic sub-populations within the urine co-existing with intracellular bacteria. UPEC invasion of the urinary bladder generates substantial changes to bladder morphology and a robust immune response. Much of our current understanding of early stages of UTI and the intracellular lifestyle is derived from studies in the mouse model (Urinary tract infections (UTIs), the second most-common cause for the prescription of antibiotics  are charse model . Examinase model . IBCs alse model . These sse model . HoweverIn addition, the bladder is an extremely complex organ; it has a stratified architecture with well-differentiated cell types; a lumen filled with urine whose composition and chemical properties can vary depending on the physiological state of the individual and is subjected to periodic and large changes in organ volume and surface area . These fOrganotypic models are well-suited to address these outstanding questions. A key strength of advanced organotypic models such as organs-on-chip  or organHere, we report the development and characterization of a bladder-chip model that mimics the bladder architecture by co-culture of a well-characterized human bladder epithelial cell line with bladder microvascular endothelial cells in a device geometry that allows the two cell types to be exposed to urine and nutritive cell culture media, respectively. The flow rates in the apical and vascular channels can be controlled independently, and multiple rounds of micturition are recreated via the application of a linear strain to the PDMS membrane that serves as the substrate for co-culture. Using this model, we show that diapedesis of neutrophils to sites of infection on the epithelial side can lead to the formation of neutrophil swarms and neutrophil extracellular traps (NETs), and that IBCs offer substantial protection to bacteria from antibiotic clearance. Our observations suggest that the role of IBCs in reseeding bladder infections upon cessation of antibiotic treatment or failure to complete a course of antibiotics might be more important than previously assumed and therefore strategies aimed toward eradication of IBCs are very crucial for treatment efficacy.We established a bladder-chip infection model by co-culturing HMVEC-Bd primary human bladder microvascular endothelial cells with the 5637 human bladder epithelial cell line  in a novel bladder-chip approach . In a smSimilarly, a majority of the epithelial cells expressed cytokeratin 7 (CK7), a general uroepithelial marker and cytokeratin 8 (CK8), a differentiated uroepithelial marker both in monoculture  and on-cfilling bladder state’), occurs through a slow and gradual increase in bladder volume due to addition of urine from kidney via the ureters, resulting in a large increase in bladder volume (filled bladder state’). Voiding of the bladder through urination rapidly reduces bladder volume, (‘voiding bladder state’) and removes the strain experienced by the bladder cells. The bladder epithelium, as a viscoelastic material, responds to the removal of strain and relaxes over the subsequent period (voided bladder state’)  .filling bladder , filled bladder , voiding bladder over a period of 2 min , and voided bladder   . We char strain) . This 6 −2) for a period of 1.5–2 hr. Low shear stress conditions have been recently reported to enhance bacterial adhesion to epithelial cells , which aids neutrophil attachment to endothelial cells  through the release of cytosolic azurophilic granules around a scaffold of decondensed chromatin . These sTo further characterize these structures, we imaged the epithelial layer of uninfected and infected bladder-chips with or without the addition of neutrophils using scanning electron microscopy (SEM). Representative images of the epithelial layers of uninfected bladder-chip controls  contrastLastly, to verify that NET formation was a direct response to UPEC and not caused by hyperactivation of neutrophils due to experimental handling, we collected the neutrophils that passed through an infected bladder-chip without attachment. Even upon infection, an overwhelming majority of the cells perfused into the chip flow out without adhering, because of the geometry of the channels in the device. NET formation in these neutrophils was not observed unless the neutrophils were themselves infected by UPEC shed from the bladder-chip . The blaUrinary tract infections that do not resolve upon intervention by the host immune system often require treatment with antibiotics. Antibiotics have complex pharmacokinetic and pharmacodynamic profiles in vivo, which we attempted to model through two successive rounds of a high dose administration of ampicillin - an antibiotic formerly prescribed for uncomplicated UTIs . Cell line tested negative for mycoplasma contamination.The 5637 human bladder epithelial carcinoma cell line  was cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS) as recommended by the supplier. Human Primary Bladder Epithelial cells  were cultured in complete epithelial cell medium supplemented with growth factors as recommended by the supplier. Human Bladder Microvascular Endothelial cells (HMVEC-Bd)  were cultured in Lonza EGM-2 MV BulletKit medium. Both the epithelial and endothelial cells were cultured in their respective medium supplemented with 1X Antibiotic-Antimycotic solution. Epithelial cells were passaged by detachment with 0.05% Trypsin at 37°C for 3–5 min followed by neutralization of trypsin with RPMI 1640 medium/10% FBS. Bladder endothelial cells were passaged and split as recommended by the supplier. The cells used in all the experiments were at ten passages or fewer. The cell lines were tested routinely for mycoplasma contamination during passaging.Escherichia coli (UPEC) strain CFT073 was originally isolated from a pyelonephritis patient (600=1.5 and corresponding to a concentration of 1.5 x 109 bacteria/ml). The bacteria were diluted 10-fold to a final concentration of (1.5 x 108 cells/ml) and resuspended in a solution of pooled human female urine  diluted 10-fold in Phosphate Buffered Saline.Uropathogenic  patient  and prov patient , as desc patient . To induBladder-chip devices made of polydimethylsiloxane (PDMS) were purchased from Emulate . The dimensions of the microfluidic device were as follows: width of the channels- 1000 μm, height of the upper channel- . For extracellular matrix (ECM) coating, a coating solution consisting of the ER-1 compound (Emulate) dissolved in ER-2 solution at 0.5 mg/ml (Emulate) was introduced in both apical and vascular channels and the chips were subsequently activated by exposing the bladder-chip for 20 min under UV light. The channels were then rinsed with fresh coating solution and the protocol was repeated once again. The channels of the bladder-chip were then washed thoroughly with PBS before incubation with an ECM solution of 150 μg/ml bovine collagen type I and 30 μg/ml fibronectin from human plasma in PBS buffered with 15 mM HEPES solution for 1–2 hr at 37°C as described previously . If not Human bladder epithelial and bladder endothelial cells were cultured inside ibidi μ−Slide eight wells for one day. The cells were subsequently fixed with 4% paraformaldehyde (PFA) for 30 min at room temperature. Fixed cells were then washed three times with 200 µl of PBS to remove residues of PFA, permeabilized with 0.15% Triton X-100 for 15 min; washed three times with 200 µl of PBS to remove residues of detergent and then incubated in a blocking solution of 1% BSA in PBS for 1 hr. The cells were then incubated with primary antibodies  at a dilution of 1:100 in an antibody incubation buffer comprising 1% BSA and 0.01% Triton-100 in PBS. The ibidi eight-wells were subsequently washed three times with PBS for 10 min each. Incubation with secondary antibody  at a concentration of 2 µg/ml in antibody incubation buffer was subsequently performed for 1 hr at room temperature. Excess antibody was removed by washing three times with PBS for 10 min each. Cell nuclei were stained with DAPI (5 µg/ml) solution in PBS for 30 min. Ibidi eight-wells were washed three times to remove unbound DAPI. Cells were covered with appropriate volume of PBS until imaging. Cells were imaged with 63X oil objective on Leica SP8 confocal microscope. Images were deconvolved using SVI Huygens .For characterization of the cell types on-chip, bladder-chips with co-culture of bladder epithelial and bladder endothelial cells were kept under pooled diluted urine and EBM2 medium using P200 pipette tips for 2 days prior to PFA fixation. The cells in the bladder-chip were then immunostained following the protocol described above. Images were acquired with Leica HC FLUOTAR 25X (NA 0.95) multi-immersion objective on Leica SP8 confocal microscope.Elveflow OB1 MK3 – Microfluidic flow control system was used to control the negative pressure applied to the human bladder-chip. The control system was connected to the compressed air line (6 bar) for the positive pressure and diaphragm vacuum pump for the negative pressure. A negative pressure (0 to −900 mbar) with a step function of −100mbar was subsequently applied to the vacuum channels in the bladder-chip using a Pressure Controller (Elveflow OB1 pressure controller). For these experiments, both bladder epithelial and endothelial cells were seeded on the respective sides in the devices. This experiment was performed with the chip maintained on the stage of the microscope and a brightfield image was acquired at each step increase of −100mbar, on Leica SP8 confocal microscope. The PDMS inter-pore-to-pore distance was measured for 14 pore-to-pore combinations at each input of negative pressure. Linear fitting was performed using GraphPad Prism (version 9).voided bladder, 0% strain) state was slowly perturbed with a linear ramp function to reach a stretched  state over a period of 2 hr. Linear strain of 10% strain was achieved by application of ca. 520–530 mbar negative pressure in the vacuum channels. This period corresponded to the filling bladder state in filled bladder, 10% strain) state over the subsequent 2 hr. Micturition or urination was recapitulated by rapidly reducing the applied strain on the bladder-chip from the stretched  state to relaxed  state over a period of 2 min. Subsequently, the bladder-chip was maintained with no negative pressure applied in the vacuum channel under relaxed  state from (4:02 hr to 6 hr). This 6 hr bladder filling and voiding cycle was repeated continuously for the rest of the experiment.The stretching of the human bladder-chip was done using the Elveflow OB1 MK3 – Microfluidic flow control system connected to the compressed air line (6 bar) for the positive pressure and diaphragm vacuum pump for the negative pressure. Human bladder-chip in the relaxed (2 (OKO labs). Time-lapse imaging was conducted using a Leica HC FLUOTAR 25X (NA 0.95) multi-immersion objective within custom made environmental chamber set at 37°C for infusion with syringe pumps. Water was pumped to the ring around the water objective at 9 Hz with pumping duration of 9 s and pumping interval 30 min, controlled by SRS software . The autofocus mode  was used to maintain the optical focus on the apical side of the PDMS membrane. The experiments were monitored frequently to ensure that the optical focus was maintained, and the experiment was halted and restarted if the focus was lost.The medium perfusion inside the bladder-chip device was achieved with the Aladdin syringe pumps. The syringes with respective media were connected to the bladder-chip via gas impermeable PharMed tubing  along with longer transparent Tygon tubings . The Pharmed tubing was connected to the inlet and outlet of the bladder-chip with 1.30 x 0.75 x 10 mm metallic tubes (Unimed) and transparent tubing with 1.00/0.75 x 20 mm metallic tubes (Unimed). The bladder-chip connected to external sources of flow was then mounted onto a 24 x 60 mm No. 1 glass coverslip for microscopy imaging. When required for stretching experiments, tubing was also connected to the stretching channels on either side of the main channel. The connected device was subsequently assembled inside a temperature-controlled microscope environmental chamber at 37°C supplemented with 5% COTo enable rapid 3-D imaging across multiple spatial locations on-chip, we utilized the capability of the white light laser on the Leica SP8 confocal microscope to image at multiple wavelengths simultaneously. The excitation wavelengths were grouped into two sequences to minimize the spectral overlap. In the first sequence, laser emission at 555 nm was used to excite the CellMask Orange stain in the bladder epithelial cells. In the second laser excitation sequence, laser emission at 500 nm and 630 nm were used to excite the YFP within the bacteria and the cytoplasmic CellTracker Deep Red in the human neutrophils. Images were acquired with a scan speed of 400–700 Hz and a zoom factor of 2.25 (206.67 µm x 206.67 µm) resulting in an XY resolution of 450 nm depending on the number of pixels acquired per field of view. Z-stacks were acquired with 1 µm step sizes. Time lapse images were acquired with interval duration of ca. 15–17 min, in a subset of experiments this was further reduced to 7.5 min by imaging with the second sequence only at a z-step size of 2 µm.The time-course of the entire experimental protocol is shown in a schematic in Stationary phase UPEC in diluted pooled urine at a concentration of 150 million cells/ml were flowed through the apical channel of the device at 1200 µl/hr for 1.5 hr. During this period, EBM2 media was flowed through the endothelial channel at 600 µl/hr.2 to enhance neutrophil attachment to endothelial cells.Next, the syringe connected to the apical channel was replaced with a fresh syringe containing pooled diluted urine, this solution was then flowed in the apical channel over the epithelial layer of the chip at a flow rate of 600 µl/hr over the next 3 hr. This allowed for the continuous removal of extracellular planktonic bacteria in the apical channel. At the same time, human neutrophils were introduced into the endothelial channel of the bladder-chip via flow. A solution of human neutrophils at a density of 2 million cells/ml isolated via negative selection was flowed through the vascular channel for 3 hr in EBM2 medium with at higher shear stress of η=1.0 dyne/cmDiapedesis of human neutrophils to epithelial side and subsequent interactions of neutrophils with UPEC was observed for the subsequent 3 hr since introduction of neutrophils into the vasculature side of the chip. During this period, the flow rate in the apical and vascular channels were maintained at 600 μl/hr and 3000 μl/hr, respectively. During this period, in a subset of experiments, only the channels that were part of the second laser scanning sequence were imaged. Images of the CellTracker Orange dye for epithelial cell identification were not acquired (first laser scanning sequence). This enabled a number of fields of view to be captured with an enhanced temporal resolution and a frame rate of up to 7.5 min. For experiments studying the formation of NETs, the experiment was halted at this stage and the infected chips were fixed, permeabilized, blocked, and immunostained with anti-myeloperoxidase (abcam) or anti-neutrophil elastase (abcam) antibodies using the procedure described earlier. For all other experiments, live imaging continued to the subsequent antibiotic treatment phase.Thereafter, syringes connected to both apical and vascular channels were changed and ampicillin at 250 µg/ml (used at ~40-fold over the minimum inhibitory concentration (MIC) of ampicillin against UPEC grown in EBM2 medium) was introduced both in the diluted urine and the EBM2 media perfused into the apical and vascular channels, respectively, for 3 hr at a flow rate of 600 µl/hour. This was the first ampicillin treatment cycle; whose purpose was to eliminate extracellular bacteria in the apical channel and allow intracellular bacterial colonies (IBCs) to be identified. The urine and EBM2 media were subsequently switched in the respective channels with antibiotic-free medium for next 8 hr to allow for IBC growth within epithelial cells. During this period, the flow within the apical channel was maintained, to remove bacteria that grew extracellularly either because they survived the antibiotic treatment or that were released from infected epithelial cells. The ampicillin and growth cycle were then repeated, to allow the assessment of the response of bacteria within IBCs to antibiotic treatment as well as characterize the subsequent regrowth.Image analysis was performed with Bitplane Imaris 9.5.1. The time-lapse imaging stack included five channels: uroepithelial cells , transmitted light (bright field channel), UPEC , neutrophils (neutrophil channel), and transmitted light (bright-field channel). Neutrophils were identified via the spot detection algorithm in Imaris used on the images from the neutrophil channel with the following parameters .Swarms of neutrophils generate dense aggregates that are ill-suited to quantification with the spot detection algorithm. To quantify the size of the swarms, we therefore segmented images in the neutrophil channel to generate surfaces via the automatic segmentation tool in Imaris with the following parameters . Unfortunately, the time resolution was insufficient to track individual neutrophils over time particularly since neutrophils formed small clumps and rapidly changed their cell shapes.For UPEC volume inside IBCs, necessary 3D volume was cropped in Imaris to ease image analysis. Total bacterial volume inside IBC was detected by creating the surface  on UPEC channel.In cases of measuring extracellular UPEC growth, surface was generated  on UPEC channel to calculate total extracellular volume in the field of view.2. Labelled human neutrophils were then washed with 10 ml of 20% FBS in RPMI phenol-red-free medium followed by centrifugation twice to remove the unbound dye. The human neutrophils were suspended in Lonza EBM2 medium at a cell density of 2 million cells/ml. In some instances, labeled human neutrophils were passed through a filter with 40 µm pores to remove neutrophil clusters that may have formed during the isolation process.Fresh human blood was procured from voluntary donations by anonymised healthy donors via the Transfusion Interregionale CRS network based in Bern, Switzerland. Donors were made aware that their donation could be used for biomedical research. No personal information about the donors was available to the end users, therefore the consent to publish could not obtained. Approval for this use of blood was provided by the Transfusion Interregionale CRS network (project number P_257). Human neutrophils were isolated via negative depletion method from human blood with MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec), following the manufacturer’s instructions. Isolation was performed without the use of a density-based centrifugation method. Isolated human neutrophils were then incubated with a 1 µM solution CellTracker Deep Red in a serum free RPMI phenol red free medium for 30 min in a cell culture incubator maintained at 37°C and 5% COUPEC within infected human bladder-chips were allowed to proliferate for 6 hr (ca. 13.5 hr from start of UPEC infection) until the end of the first IBC growth cycle. After 6 hr, bladder-chips were fixed at room temperature for 1 hr with a mix of 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The fixed bladder-chips were kept in the fixative overnight (at 4°C). Post overnight fixation, bladder-chips were cut open from the apical channel side. A scalpel was used to cut approximately in the middle of the apical channel side (height=1mm) to expose the bladder epithelial cells. The fixed chips were further fixed for 30 min in 1% osmium tetroxide in 0.1 M cacodylate buffer followed by washing with the distilled water. Next, the bladder-chips were dehydrated in a graded alcohol series and dried by passing them through the supercritical point of carbon dioxide . Finally, the bladder-chip was attached to an adhesive conductive surface followed by coating with a 3–4 nm thick layer of gold palladium metal . Images of the cells were captured using a field emission scanning electron microscope .Uninfected bladder-chip controls were fixed at the same time point (ca. 13.5 hr from start of the experiment). For the case of NETs formed on epithelial layer, an uninfected human bladder-chip was fixed 2 hr after the introduction of neutrophils into the vascular channel of the chip.UPEC within infected human bladder-chips were allowed to proliferate for 6 hr (ca. 13.5 hr from start of UPEC infection) during the first IBC growth cycle. After 6 hr, bladder-chips were fixed at room temperature for 1 hr with a mix of 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The fixed bladder-chips were kept in the fixative overnight (at 4°C). Post overnight fixation, bladder-chips were cut open from the apical channel side. A scalpel was used to cut approximately in the middle of the apical channel side (height-1mm) to expose the bladder epithelial cells. The bladder-chips were then washed in cacodylate buffer , postfixed for 40 min in 1.0% osmium tetroxide with 1.5% potassium ferrocyanide, and then fixed again with 1.0% osmium tetroxide alone. The bladder-chips were finally stained for 30 min in 1% uranyl acetate in water before being dehydrated through increasing concentrations of alcohol and then embedded in Durcupan ACM  resin. The bladder-chips were then placed in Petri dishes so that approximately 1 mm of resin remained above the cells, and the dish than left in an oven at 65°C for 24 hr. Regions on interest, and corresponding to structures imaged with light microscopy were trimmed from the rest of the device, once the resin had hardened, and thin, 50 nm-thick sections were cut with a diamond knife, and collected onto single-slot copper grids with a pioloform support film. These were contrasted with lead citrate and uranyl acetate, and images taken with a transmission electron microscope at 80 kV . public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:Reviewers value the development and characterization of a bladder-on-chip infection model for recapitulating the multiples factors involved in UPEC driven UTIs. Notably, it consists of human bladder epithelial cells (cell line or primary), bladder microvascular endothelial cells, neutrophils and urine that are also subjected to mechanical changes mimicking those occurring during bladder filling and micturition. This model is a lot more complex than in vitro tissue culture models and more amenable to analysis such as imaging than animal models and therefore constitute a distinct advance for in vitro modeling of UTI that has potential to reveal key aspects of UTIs and reasons for the difficulty to clear these infections with antibiotics.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Milica Radisic (Reviewer #2).Thank you for submitting your article "Dynamic persistence of UPEC intracellular bacterial communities in a human bladder-chip model of urinary tract infection" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential Revisions:A few points need addressing to strengthen the study:– A limitation of the work represents the use of an epithelial cell line. Is it possible to repeat some aspects of this work with a primary epithelial cells for a greater relevance?– Please repeat the experiment with at least another AB that can reach the intracellular bacteria, since ampicillin will not.– Reword the paper to avoid overinterpretation of the data in paying particular attention to avoid the following:1. The immediate interpretation of all intracellular structures as IBCs. IBCs were first described in mice and IBCs usually refer to a specific structure that is formed (at least) in mice and humans in vivo. Similar structures previously observed in vitro are rightfully carefully described such as "structures resembling IBCs". Please use similar care here.2. The immediate interpretation of all data in Figure 2 as neutrophil swarms and NETs.3. The claim that a 10% linear change in dimension is "physiologically relevant" and "a significant proportion" of that seen in vivo.There are occasional sentences where the appropriate care is taken but this is interspersed with writing that is assuming the point is already proven . Essential Revisions (for the authors):A few points need addressing to strengthen the study:- A limitation of the work represents the use of an epithelial cell line. Is it possible to repeat some aspects of this work with a primary epithelial cells for a greater relevance?We agree with the Reviewers in that a primary epithelial cell model would be a closer mimic to the in vivo physiology. However, a complete set of experiments with primary cells is beyond the scope of the manuscript. We have therefore repeated certain key experiments with primary cells pertaining to the formation of IBCs and the subsequent treatment with fosfomycin (related to point 2). Owing to the constraints imposed by the COVID-19 pandemic, these experiments were limited to snapshots taken at critical timepoints in the antibiotic treatment cycle. In the revised manuscript, we provide clear evidence of the formation of numerous IBCs in bladder-chips reconstituted with primary cells , as well as snapshot evidence of regrowth despite two rounds of fosfomycin treatment . These results reinforce the strength of our data with the cell line and ampicillin.- Please repeat the experiment with at least another AB that can reach the intracellular bacteria, since ampicillin will not.et al., PMID: 11713400). Our choice for the use of ampicillin was also motivated by the fact that this antibiotic has been used extensively in laboratory experiments with bacteria under axenic conditions. However, we do agree that current first-line therapeutics such as fosfomycin have an increased cell penetrability and a different bacteriocidal mechanism. We have therefore repeated the antibiotic treatment experiments with fosfomycin for both primary cells (snapshots only) and the 5637-cell line (lower resolution imaging to provide higher throughput) and included this data in the revised manuscript. We observe IBCs both in 5637 cells  as well as primary human bladder epithelial cells . Although fosfomycin reduces IBC volume by a significantly larger fraction than ampicillin  we nevertheless observe regrowth from IBCs through two rounds of treatment . This data significantly strengthens our overall conclusion that IBCs may be a protective niche against antibiotic treatment for a prolonged period of time.We disagree somewhat with the reviewers in their categorical statement that ampicillin does not penetrate host cells; there are studies that show host cell penetration and activity against a range of pathogens . Ampicillin was prescribed for urinary tract infections prior to the widespread development of antibiotic resistance  in mice and humans in vivo. Similar structures previously observed in vitro are rightfully carefully described such as "structures resembling IBCs". Please use similar care here.We agree with the reviewer that although certain aspects of the structures we observe conform to established descriptions of IBCs, they may differ from IBCs formed in infected animals in other respects. In the Revised Manuscript between Lines 331 – 358 we have altered the flow of the narrative to first describe the morphological characteristics of these structures before assigning them as IBCs. Furthermore, we have therefore inserted the following sentence at Lines 347-349:“Hereafter, we shall refer to these IBC-like structures as IBCs for simplicity, acknowledging that these structures may differ from IBCs reported in infected animals.”2. The immediate interpretation of all data in Figure 2 as neutrophil swarms and NETs.We agree with the reviewer that our interpretation in the text of the data in Figure 2 was premature. In the Revised Manuscript, we have edited the text between Lines 271 – 310 at numerous places to describe the evidence from the immunostaining experiments and SEM images first before classifying the bundles that form between neutrophils as NETs.3. The claim that a 10% linear change in dimension is "physiologically relevant" and "a significant proportion" of that seen in vivo.We have edited the Revised Manuscript to remove the two descriptors. In the revised manuscript, at Line 171-172, the bladder-chip model is described as:“with an applied strain that partly mimics the physiology of bladder filling and voiding cycles”Furthermore, in the Revised Manuscript at Lines 163-165, we have clarified that the applied strain of 10% is a small fraction of the maximum applied strain in the bladder.“We therefore limited the linear strain applied to a maximum of 10%, which is of the same order of magnitude, albeit a small fraction of the typical strain experienced by the bladder tissue in vivo.”There are occasional sentences where the appropriate care is taken but this is interspersed with writing that is assuming the point is already proven .We apologies for the error on our part. We thank the reviewers for pointing out these specific instances and we have edited these sentences as part of our response to the earlier points so that the lines of evidence are listed first before the conclusion is drawn. The instances pointed out by the Reviewers at lines 477-489 and 481-483 corresponded to text in the Discussion which, in the original manuscript, partly repeated evidence already presented in the Results section. For ease of reading, in the revised manuscript we have edited these lines to avoid referring to the primary data. We hope that with the revised text in the Results section, the reviewers and readers are sufficiently convinced about the strength of our findings that we do not need to present the evidence again in the Discussion.


This cross-sectional study assesses age and sex differences in risk of 1-year mortality in patients with emergency department visits due to alcohol compared with the general population in Ontario, Canada. We evaluated the probability of death in the year after 1 or more alcohol-related ED visits and the differences by age and sex.In the past 2 decades, alcohol-related emergency department (ED) visits have increased in Canada and the US.STROBE) reporting guideline.This repeated cross-sectional study identified all ED visits due to alcohol and deaths from any cause for individuals aged 15 to 59 years in Ontario, Canada, between January 2003 and December 2017 using linked health administrative data through ICES. This study was approved by the privacy and legal offices of ICES and followed the Strengthening the Reporting of Observational Studies in Epidemiology  of 1, 2, or 3 or more ED visits due to alcohol in the year after the initial visit. Each individual could contribute 1 year of follow-up, which began after their first point of eligibility , of which 295 011 individuals  had 1 or more ED visits due to alcohol . The perOf the individuals with 1 or more ED visits due to alcohol, 265 398 (90%) had a single ED visit within 1 year. Furthermore, 4310 (73.8%) deaths occurred among individuals with a single alcohol-related ED visit.The findings of this study suggest a substantial elevation in the risk of mortality for individuals after 1 or more ED visits due to alcohol. The findings also suggest that a single ED visit due to alcohol, even in younger individuals, was associated with a substantial elevation in the risk of death and that most deaths were associated with a single visit.4 Studies suggest that improving uptake of brief alcohol interventions delivered in the ED and increasing access to medical services for addiction may reduce harm associated with use of alcohol.6A study limitation was the inability to identify the role of alcohol in each death. Whereas a single visit may be dismissed as a unique event, and patients may receive minimal follow-up after discharge from the ED, our data suggest that any ED visit due to alcohol is associated with future adverse events. Increases in delivery of interventions may be warranted because ED visits and deaths due to alcohol have been increasing in North America.


This study aimed to develop up-to-date spatial distribution visualisations of the K13 markers of artemisinin resistance from patients' samples in Asia and articles already included in the WWARN database were excluded. Data were extracted from the published articles and authors were contacted when information was missing. We used the lowest administrative unit levels for the sampling locations of all the K13 data to describe the spatiotemporal distribution. The numbers of samples tested and those with each molecular marker in each administrative unit level were aggregated by year to calculate the marker prevalence over time.In this systematic review and spatiotemporal analysis we used the WorldWide Antimalarial Resistance Network (WWARN) surveyor molecular markers of artemisinin resistance database. We updated the database by searching PubMed and SCOPUS for studies published between Jan 1, 1990, and March 31, 2021. Articles were included if they contained data on K13 markers from 16 613 blood samples collected from 1991 to 2020 from 18 countries. Most samples were from Myanmar (3842 [23·1%]), Cambodia (3804 [22·9%]), and Vietnam (2663 [16·0%]). The median time between data collection and publication was 3·6 years . There was a steady increase in the prevalence of WHO-validated K13 markers, with the lowest of 4·3% in 2005 (n=47) and the highest of 62·9% in 2018 (n=264). Overall, the prevalence of Cys580Tyr mutation increased from 48·9% in 2002 to 84·9% in 2018.Data were collated from 72 studies comprising From 2002 to 2018, there has been a steady increase in geographical locations and the proportion of infected people with validated artemisinin resistance markers. More consistent data collection, over more extended periods in the same areas with the rapid sharing of data are needed to map the spread and evolution of resistance to better inform policy decisions. Data in the literature are reported in a heterogeneous way leading to difficulties in pooling and interpretation. We propose here a tool with a set of minimum criteria for reporting future studies.This research was funded in part by the Wellcome Trust. Malaria has been declining globally with a 50% reduction of reported cases and an 84% reduction in deaths during the Millennium Development Goals era (2000–15); however, no significant progress in reducing the number of global malaria cases has been made since 2015. Malaria case incidence declined by 27% between 2000 and 2015 and has been increasing since 2015.7kelch13 (K13) gene associated with artemisinin resistance (with the phenotype of slower parasite clearance) in 2014 has enabled monitoring for artemisinin resistance in research studies and increasingly in routine surveillance.K13-propeller domain mutation present.K13 mutations over a large area. Such results can then be used to inform therapeutic efficacy study site selection or trigger a further investigation for studies to identify the resistant phenotype.Effectively managing artemisinin resistance requires tools to monitor its spread over time and space to guide interventions to control or ideally eliminate resistant parasites.Evidence before this studykelch13 (K13) markers and aggregated K13 markers per country. Additionally, the WorldWide Antimalarial Resistance Network (WWARN) K13 surveyor charts K13 global maps with study sites drawn as points using an individual patient data meta-analysis classification. However, no study has aggregated K13 marker data by provinces or district in a policy maker-friendly format and examined their temporal change in Asia. By March 1, 2021, no such study was obtained from the WWARN database, and from searching PubMed and Scopus databases using the search terms “(artemisinin OR kelch OR kelch13 OR k13) AND (Asia)”.In 2014, a multicentre study showed the worldwide map of Added value of this studyK13 markers and the first study to aggregate all published K13 marker data in Asia and to map them by province and district. In this study we also identify that non-uniform, patchy, and delayed reporting are crucial challenges in K13 surveillance. To optimise K13 surveillance, this study proposes a new tool for assessing the minimal essential information to assist in pooling K13 marker data from different studies.This is the first study to use the 2020 revised WHO classification for Implications of all the available evidenceThere is a steady increase in geographical locations and the proportion of malaria infected people with validated artemisinin resistance markers. However, inconsistent data collection and delayed publication hamper our knowledge of the current status, with the available data providing a snapshot of the situation up to four years ago. More consistent and rapid sharing of data from the same areas are needed to map the spread and evolution of resistance to inform policy decisions better.There is a range of current initiatives monitoring antimalarial resistance using this method, with the main output being maps of resistance marker prevalence.19K13 markers, then present this evidence of artemisinin-resistant Plasmodium falciparum malaria in a policy maker-friendly format to help guide the planning of malaria control and elimination strategies.This study aimed to determine the spatial and temporal distribution of the genetic markers of artemisinin resistance in Asia using data from the published literature on This study used a sequential mixed-methods design; here, we present the quantitative part that includes a systematic review and descriptive cross-sectional spatiotemporal analysis. The following part used a qualitative end-user usability assessment that comprised interviews and feedback from national malaria programme staff to optimise map presentation for policy makers, which will be published separately. Therefore, this Article presents only the spatiotemporal analysis findings and maps with optimised formats.The previously established database of the molecular markers of artemisinin resistance in the WorldWide Antimalarial Resistance Network (WWARN) surveyor  was combined with a systematic review of all published research papers in PubMed and Scopus using the search terms “artemisinin”, “kelch”, “kelch13”, and “k13” along with geographical terms as shown in K13 markers of artemisinin resistance from patients' samples), and, if eligible, were included for data extraction into an updated K13 marker dataset. In case of conflicts a third reviewer (KIB) was invited to review and their decision would be final.Citations were downloaded and screened using Mendeley citation software (version 1.19.4). R software (version 3.6), stringr, and tidytext libraries were used to scan the article titles and abstracts to remove duplicates, non-malaria, non-artemisinin related articles, studies that were not using blood samples taken from patients, and citations that were already contained in the WWARN database.K13 molecular markers; this included whether K13 markers were present or absent and the total number of tested samples. The numbers of samples tested and those with each molecular marker in each subnational level were aggregated by year to calculate the marker prevalence over time. The month and year of sample collection were those reported as the end of sample collection for that administrative unit in the relevant paper where this was available (n=47). For those studies without this administrative unit's information, we used the month of the end of the sample collection period (n=11). For those where only the year was provided, we assumed the month to be December (n=8) to give the most conservative (shortest) estimates of the time from sample collection to publication. Validity and risk of bias between studies and in overall data extracted were mitigated by adhering to the study methods .We examined the full texts of the obtained articles to identify the lowest administrative unit levels for the sampling locations. For the papers with this information missing (n=2) we contacted the authors by email to request these details. Where information was provided on imported cases, the probable location of transmission was used. This process made it possible to aggregate data from multiple studies at least at administrative level 1 (provinces or regions) for all countries. We took a consensus decision to aggregate data to a level that can achieve geographically comparable sizes of subnational administrative unit levels between countries. Therefore, studies in China, India, Pakistan, and Myanmar (with large administrative level 1 units) had data aggregated at administrative level 2 . In each study and year of data collection, we identified and extracted all evaluated K13 markers.The final dataset of aggregated molecular marker prevalence was imported for curation into the RStudio development environment  and geocoded to match The Database of Global Administrative Areas (GADM version 3.6).K13 mutations by their level of evidence for P falciparum artemisinin resistance using the WHO classification (2020), with the categories of WHO validated or confirmed, WHO associated or candidate, WWARN associated, not associated, and wild type  and 20 were eligible for analysis along with 52 articles in the WWARN database  and 2013 (2384 [14·4%]), and few samples were included from before 2009 . The samThe median number of samples collected per administrative unit for all years combined was 42 (range 1–2052), with 40 (32%) administrative units having less than 20 samples and 33 administrative units (26·4%) having more than 100 samples.K13 marker in 2014.The median time from sample collection to publication by the administrative unit was 3·6 years  in northeast Thailand, western Cambodia, and central Vietnam. Only associated markers were found in western Myanmar.Our study found that samples from the GMS were collected in more years than outside the GMS . The GMSOf the 60 administrative units with data in the GMS, 41 (68%) had data from multiple years . The preDisaggregated data by country showed Cambodia, Laos, Thailand, and Vietnam to have a clear trend of increased validated markers over time ; video. . We observed the highest prevalence of molecular markers of artemisinin resistance in the eastern parts of the GMS , Changlang , and Kolkata . In 2016 and 2019, only K13 wild-type parasites were reported from 16 districts in India, and in Afghanistan, Bangladesh, and Nepal.There was a sparse distribution of artemisinin resistance data in India, Afghanistan, Bangladesh, Nepal, and Pakistan . AlthougOverall, we found an increase in the prevalence of WHO-validated and WHO-associated markers from a mean of 18·6% for samples collected before 2011  to a mean of 52·9% for 2011–18 . Before 2019, the Cys580Tyr marker was detected every year, except in 2003 and 2005 . Some wiP falciparum artemisinin resistance markers (K13 mutations) over time in Asia, including the most recent published data and a subset of unpublished data . To do this analysis, we searched citations from the PubMed database and Scopus to update the WWARN K13 dataset. We added additional spatial and temporal details to produce a dataset of K13 markers containing 72 citations. The P falciparum K13 propeller mutations were used to approximate the spatiotemporal distribution of artemisinin resistance, with an increase in resistance markers by type and geographical extent shown in a range of thematic maps. Over time, we observed a consistent reporting of an increase in the prevalence of artemisinin resistance validated and associated markers in Asia. These mutations were first identified along the border between Cambodia and Thailand in the Battambang and Pailin provinces.P falciparum malaria is most prevalent in these countries.We have presented a description of all currently available information on changes in the geographical distribution of genetic markers of P falciparum, which resulted in a significant decrease in cases.K13 markers that are known to be associated with markedly slower parasite clearance and of markers of resistance to artemisinin and artemisinin-based combination therapy partner drugs.In the present study, we found that although the Cys580Tyr mutation occurs in all the GMS countries, the most frequent validated mutations in the eastern GMS  were Cys580Tyr, Arg539Thr, Tyr493His, and Ile543Thr. In western GMS , the most prevalent validated mutations were Phe446Ile, Asn458Tyr, Pro574Leu, and Arg561His. This distribution is consistent with previously published studies, showing localisation of mutations in specific geographical areas, such as Asn458Tyr and Arg561His in the eastern and northern GMS, Arg539Thr in southern and east GMS, and Ile543Thr and Tyr493His in east GMS.K13 marker in 2014. For studies only analysing samples collected after the discovery of K13, the lag was still substantial at 2·6 years, despite using conservative assumptions for studies in which detailed time information was not available. This lag limits the usefulness and relevance of this marker data for guiding policy decisions. The relative lack of published studies reporting the prevalence of K13 markers in samples collected in 2019 and 2020 is likely to be due to this lag between sample collection and publication. The absence of validated or associated K13 markers in those years is therefore a consequence of the published data being from only a few sites in Pakistan, India, and Saudi Arabia. Large scale sample collections have been ongoing in the GMS during these years, which will contribute more K13 distribution data in the near future.Our analysis found a long lag time between sample collection and publication (median 3·6 years). Part of this lag was due to many studies retrospectively analysed samples that had already been collected before the discovery of the K13 mutations were investigated (and which were not) and when and where the samples were collected. This disparity required us to either make assumptions or constrain the possible spatial and temporal resolution of the analysis. Because of these limitations, we propose a set of reporting criteria for studies that collect K13 molecular marker data, and we have developed a tool that can be used to collate data from published studies and generate a score for these minimal criteria  with data. Although the K13 propeller mutations to describe artemisinin resistance; however, a 2017 study suggests other K13 mutations apart from propeller mutations  might also be associated with artemisinin resistance.K13 propeller mutations remain the preferred marker for surveillance of P falciparum artemisinin resistance. Our study found a considerable number of unevaluated SNPs that have not yet been classified or graded by WHO or included in the WWARN individual patient data meta-analysis. Such markers could potentially influence the artemisinin efficacy in the geographical areas analysed in this study. Delays in evaluating the association of molecular markers with delayed parasite clearance contribute to the increase of unevaluated SNPs; to date there has been a 3-year gap between WHO evaluations  and only a single evaluation by WWARN in 2017.Prompt reporting of routine surveillance or research activities with a consistent, repeated collection of molecular markers in the exact location (sentinel sites) would allow for more accurate and informative spatial and temporal analysis. The present study's findings can help identify those areas that would be most suitable for such surveillance activities. However, unless sentinel sites have broad coverage, detecting the emergence of resistance might also be delayed. This study used the This study combined data from all available published sources to define the increase in prevalence and geographical extent of artemisinin resistance markers in Asia. More consistent data collection is needed from the exact locations, from a wider geographical area and over more extended periods to map the spread and evolution of resistance as it continues to unfold. This study also highlights the need for more rapid dissemination of molecular marker data so it is available in time to guide policy decisions.https://www.wwarn.org/tracking-resistance/artemisinin-molecular-surveyor).The complete and updated datasets, plots, and maps generated and analysed during this study are publicly available on the WWARN website (We declare no competing interests.


Implementing machine learning methods into the RNA-seq data analysis pipelines can further improve the efficiency of data utilization in clinical decision making. In this article, we present how machine learning methods can be used to go one step further in data analysis of the global gene expression datasets, namely, to develop models that are able to classify individual cancer samples based on well characterized reference samples. We used the publicly available endometrial cancer sample RNA-seq datasets of the TCGA project to develop a model that can separate G1 and G3 cancer samples with an accuracy of 85%. Our model could also further stratify G2 samples into high-risk and low-risk subgroups. Moreover, with an iterative retraining approach, we could subselect twelve genes that performed similarly in the stratification. Our results were validated by the survival data of the patients.The tumor grade of endometrioid endometrial cancer is used as an independent marker of prognosis and a key component in clinical decision making. It is reported that between grades 1 and 3, however, the intermediate grade 2 carries limited information; thus, patients with grade 2 tumors are at risk of both under- and overtreatment. We used RNA-sequencing data from the TCGA project and machine learning to develop a model which can correctly classify grade 1 and grade 3 samples. We used the trained model on grade 2 patients to subdivide them into low-risk and high-risk groups. With iterative retraining, we selected the most relevant 12 transcripts to build a simplified model without losing accuracy. Both models had a high AUC of 0.93. In both cases, there was a significant difference in the relapse-free survivals of the newly identified grade 2 subgroups. Both models could identify grade 2 patients that have a higher risk of relapse. Our approach overcomes the subjective components of the histological evaluation. The developed method can be automated to perform a prescreening of the samples before a final decision is made by pathologists. Our translational approach based on machine learning methods could allow for better therapeutic planning for grade 2 endometrial cancer patients. Endometrial carcinoma represents the fourth most frequent type of malignancy among women in developed countries . Based oThe tumor grade serves as an independent marker of survival  and may In recent years a growing number of approaches have emerged for the better characterization of cancers, which are based on the molecular profiling of tumors. The Cancer Genome Atlas (TCGA) project is one such outstanding example that performed detailed molecular profiling of tumors from copy number variation and mutation analysis, through RNA-Seq based gene expression and miRNA profiles. A direct result of the TCGA project is the identification of four molecular prognostic subgroups within endometrial tumors . This coOur objective was to divide G2 patients into high-risk and low-risk subgroups based on their gene expression profiles. Such a gene expression-based stratification approach for endometrial cancer could complement the current histological classification with an objective, semi-automated component that could reduce uncertainties in the therapeutic decision-making for such patients. In order to exploit the full benefit of global gene expression datasets, we decided to complement the gene expression data analysis methods with machine-learning classification tools. Our approach is founded on the objective laboratory procedure of RNA sequencing . Our metRecent developments in RNA-based diagnostics make the implementation of RNA-based screens feasible by RT-QPCR from as small biological sample as a needle biopsy provides. As demonstrated by Visser et al. , the besAccording to our knowledge, this is the first report that proposes a shortlist of gene expression-based biomarkers that could help to make the transition from a three-level grading system to a binary one. Such a development is in line with the objectives of the clinical community as proposed by Soslow et al.  and withThe current 3-tier grading system of endometrioid carcinoma is not ideal for patients included in the grade 2 group as the result could be either under- or overtreatment.An objective, reproducible gene expression based method could separate patients included currently in grade 2 class into low-risk and high risk subgroups.We have developped a machine learning classifier method that can stratify grade 2 patients in high-risk and low risk subgroups.For translational purposes we refined this method to perform the stratification of patients with simillar performance using as few as 12 biomarker genes.Based on recent developments in RNA-based diagnostics, translation into clinical practice of our proposed biomarkers could contribute to the development of a robust, binary grade classification system by which more reliable preoperative classification could be implemented.The above mentioned challenges of our work and our contributions to the field may be summarized as follows:https://bioinformatics.mdanderson.org/public-software/tcga-batch-effects/ accessed on 30 October 2020). The extent of the batch effect was not found to be significant (DSC < 0.5), thus eliminating worry about any disturbing impact. The clinical data of the 546 patients of the samples were downloaded from www.cbioportal.org (accessed on 28 October 2020).During our work we used R 3.6.1 and Python 3.6.5 programming environment, relying on their basic functions, as well as specific packages and modules. The machine-learning model and the simplified model using the minimally required number of genes were developed in Python, while R was used for the remainder of the analysis steps. Our samples were derived from the TCGA endometrial carcinoma study . Using tThe samples that were inadequate for our purposes based on clinical parameters were screened using the dplyr 1.0.3 package. Based on our applied conditions, we continued working with only those patients who had not received neoadjuvant therapy prior to the sample collection as neoadjuvant therapy could influence the gene expression profile . A requiThe RNA-sequencing data studying 60,488 transcripts per sample were processed using the DESeq2 1.24.0 package . . The risk ratio of the Cox model was calculated at 2.54 , therefore, our model was able to identify a major portion of high-risk patients from the perspective of relapse.In order to separate G2 samples into low-risk and high-risk subgroups, we applied the model (developed in the previous stage) to the gene expression data of the G2 samples. Using the trained model, the G2 samples could be divided into low-risk and high-risk groups with 62 and 49 samples in the respective subgroups. In order to validate our approach, we compared the relapse-free survival (RFS) of the identified subgroups. As shown in At this point, we were interested in whether a similar accuracy could be achieved while using a smaller number of genes than the 24,349 transcripts. For this purpose, we used the G1 and G3 datasets and arranged the transcripts in descending order based on their contribution to overall variability, represented by the absolute value of their average coefficients in the outer loop, and then repeated the former model development iteratively. Considering the average AUC values calculated by this method and the AUC values in the test set, we selected the 12 most important genes for our simplified model because it provides an easy-to-use number for a qPCR-based gene expression test  [We further validated each of these 12 genes individually by using st 2021) ,30, an oOur classification model used the machine-learning algorithm to successfully identify the gene expression profiles of G1 and G3 patients and the differences between the groups. Based on this, the trained model was able to identify the high-risk G2 patients, thus complementing the current histological classification. Our simplified model with 12 genes produced similarly good results. The latter may be suitable for further development as a qPCR-based clinical test that is significantly cheaper and faster than a global transcriptome approach. At the same time, RNA isolation and qPCR itself are processes that may be highly automated ,21. ThesOf the 12 genes used in the simplified model, some of the protein-coding genes are known to have a role in the pathogenesis of endometrial carcinoma, while for the time being other genes have been studied in detail but only in tumor types other than endometrial carcinoma. We found that high expression of FOXB1, HABP2, EDN3, B3GAT1-DT, and DKK4 was associated with low-risk phenotype, while high expression of RPL41P1, MAL, UCHL1, CRABP1, PEG10, RPS28P7, and MLF1 was responsible for more clinically aggressive behavior.Similar to the other Dickkopf proteins, DKK4 controls the WNT signaling pathway ; its difBesides the development of the qPCR-based clinical decision support system, further study of these genes may be justified because they provide a great amount of useful information on the molecular characteristics of endometrial carcinoma, and the products of these genes may even serve as potential drug targets. As some non-coding RNA molecules can be identified from these regions, further studies are needed to gain a better understanding of these non-coding RNA molecules.As a potential future development, the immunohistochemical investigation of our proposed biomarkers could be considered. Two of our biomarkers that are present on the RNA level in a variety of databases were earlier classified as pseudogenes; therefore a limited number of prior data is available. On the other hand, our proposed gene list can provide good performance  even with three genes therefore immunohistochemical measurements should be considered at a later stage for at least some of the genes.The goal of the TCGA project was to generate comprehensive molecular maps of a large variety of cancer types. In the project 33 cancer types were included ,46. The Another important aspect that should be considered if we investigate the potential limitations of our study is that the datasets used in our investigations, generated in the TCGA Project, were generated from samples that contained not only cancerous cells but very likely normal cells, necrotic cells, and all other cell types characteristic of cancer tissues. Therefore, the generated molecular maps do not show only the profiles of the cancer cells but the average of a variety of cells present in the processed bio-samples. While this is a limitation from a basic science point of view, it can be a benefit for a translational approach that aims to identify the most robust biomarkers present in a cancer sample.Overall, our results and the additional research that can follow up our results could help to further improve the living prospects of women suffering from endometrioid endometrial carcinoma. By further stratifying grade 2 patients into high-risk ad low-risk subgroups our development can contribute to the implementation of a binary FIGO grading proposed by Soslow et al.  and bettSpeeding up the translation of basic scientific results into clinical practice requires novel methods that can provide reliable pre-screening information for the pathologist. Global gene expression analysis performed with RNA-seq became technically available in every major medical center; still, these methods are not used routinely in medical decision making due to the complexity of the generated data. Implementing machine learning methods into the data analysis pipelines can speed up the final clinical decision-making and thereby, reduce the waiting times of the patients for a final diagnosis.While machine learning methods have been implemented in automated image analysis and have significantly improved the quality of clinical decisions, we observe a delay in their implementation into nucleic acid based clinical decisions. In this article, we present a machine learning based method that can stratify the G2 endometrioid cancer patients into low-risk and high-risk subgroups based on their gene expression profiles. With iterative retraining, we have developed a model that relies on only a set of twelve genes and has 84% accuracy. Our approach could be used to generate semi-automated prescreening reports and therefore speed up the final clinical decision-making by the pathologist.


Sexual education has become increasingly important as unhealthy sexual practices and subsequent health risks become more prevalent during adolescence. Traditional sex education teaching methodologies are limiting for digital natives exposed to various digital technologies. Harnessing the power of technology applications attractive to the younger generation may be a useful approach for teaching sex education.The aim of this study was to improve sexual health knowledge and understanding of the problems associated with unhealthy sexual practices and address sexual and reproductive health challenges experienced in a low-tech setting.A participatory design approach was used to develop the digital gamified methodology. A sample of 120 secondary school students aged 11-15 were randomly assigned to either experimental or control group for each of the 3 teaching approaches: (1) gamified instruction ; (2) gamification ; and (3) traditional teaching (TT) methods.119=41.252; P<.001 [2 tailed]). Analysis of variance indicated no significant differences across groups for pretest scores . Significant differences across groups were evident in the posttest scores. Students in the SG and GM groups had higher average scores than the TT group . Students reported increased learning motivation, attitude, know-how, and participation in learning (P<.001) when using SG and GM approaches.The SG and GM approaches were more effective than TT methods in teaching sexual health education. Specifically, the average scores across groups demonstrated an increase of mean scores from the pre- to posttest  may improve sexual health education. These findings may also be applied by practitioners in health care settings and by researchers wishing to further the development of sex education. Unhealthy sexual activity and its related diseases have increased globally. Nowhere is the effect of sexually transmitted diseases more apparent than in the countries of sub-Saharan Africa (SSA). In SSA countries, many adolescents are exposed early to sexual intercourse and sexual and emotional abuse . ConsequDigital health games designed to target sexual health practices have increasingly demonstrated their capabilities, appeal, and influence on educating digital native adolescents . GamifieDigital games facilitate role playing and offer challenging approaches to learning improving attitude and decision-making skills applicable to real-life scenarios. Because digital platforms offer an engaging approach for learners, they promote knowledge acquisition . The novThe aims of this study are to (1) add to the limited existing knowledge of game-based technologies and (2) address the interest in using this novel kind of technology as the teaching approach in a low-tech setting in Africa. First, we hypothesized that the application of game elements and mechanics in learning would enhance the sexual health literacy of teenage students. Second, we hypothesized that the teenage students would develop an attitude toward gamified instruction (SG and GM) that was more favorable and receptive than that toward the traditional learning approaches. Henceforth, this study looks at how gamified instruction can improve the sexual health education of adolescents, address their sexual health challenges, and help them overcome those challenges, all of this in developing countries, which tend to be far less invested in digital technologies than developed countries .This study employed a quasi-experimental research design. The design guided the sexual health literacy interventions for students clustered in 3 classes. Sexual health education was made mandatory for all students to sanction the randomization technique ,27. The The study involved teenage students (n=120) aged 11-15 who were enrolled in a secondary school at the time of the study. The research was carried out in a school in Dar es Salaam, Tanzania. This school was selected because it had 2 computer laboratories, each able to accommodate around 40 students. They had internet connectivity, a power supply, and a standby generator. The 3 classes had around 40 students each. Everyone in a class was in the same grade. The participants were not chosen randomly. Each participant was assigned to either an experimental group or a control group based on their intact classroom settings. The researchers had no authority to form or annihilate the existing study population setting. The research team randomly designated 2 of the classes as experimental  and 1 class as control .Each group was unaware of the other groups. The learning materials were the same for all the 3 groups. The only differences were in the instructional approaches. The participants were from different schools that had taken part in the revision and participatory design process of the interventions. Finally, there were 5 topics, each covered in one 40-minute class per week, as reported in SG and GM interventions were developed following “activity theory” , “designStudents assigned to receive TT were treated as the control group. They were taught in a conventional classroom manner. Their teacher taught 1 day a week for 40 minutes for 5 weeks. Students were given hand-outs for further reading after each session. No digital technology was used.Students in this group received sexual health education using an SG approach  and 3. AThe game has an introduction which presents the general learning objective. Each topic has a game scenario in which there are avatars representing a teacher and students interacting in a classroom. There were conversations between the teacher and the students’ avatars. After watching the scenarios, the students were asked to complete the quizzes online. There were 10 quizzes on each topic, to be completed within 90 minutes. Scores were provided for correct answers, and students would lose points for each wrong answer. The students also had an opportunity to repeat the gameplay within the 40-minute margin.GM is the process of giving some of the characteristics of real games to activities that are not games. GM aims to make the learning activities more interactive. This is supposed to motivate students to learn in a way that is more effective because it is fun. GM is an emerging technique within education ,34,35. TThere were 5 topics and for each topic 40 minutes were allocated per week for 5 consecutive weeks. Each student studied individually, but they could interact online via a discussion forum devoted to each topic. Students were asked to read and practice the lesson materials provided online. There were 10 questions under each section, some true/false and some multiple choice. There were 7 types of badges . One wasDuring the design of the interventions the selected research team and participating parties, especially the teachers and sexual/reproductive health specialists, were involved in developing a set of questions covering the 5 topics taught. This test was titled Adolescent Sexual Health Literacy Test (ASHLT). There were 50 questions, 10 per topic in the following format: Section A (multiple choice), Section B , and Section C (short answer). The ASHLT took up to 45 minutes to complete. Before initiating the actual learning, students were asked to do a pretest (using the ASHLT) to assess their sexual health knowledge (baseline). Within a week following the training, the same ASHLT questions were given to measure their level of understanding.motivate students, improve their attitude, increase their knowledge acquisition, and increase their engagement in learning. Several scholars have employed the method for evaluating the efficacy of the 4 components of MAKE independently  in a competitive [way], which helped me gain problem solving-skillsGM-7Students also pointed out that...the learning inspired me; hence I focused on learningSG-2...I was extremely interested in the learning approachGM-4...the availability of badges encouraged [me to learn] the subjectGM-2By contrast, students from the TT group were largely negative about their learning experience:...there were no visualsTT-5...[there was] no clarification on many issuesTT-2...[there were] limited, or no activities for concentrationTT-6...little or none of the critical thinking strategies were provided, including role play, demos, quizzes, team-work activities, [or] collaboration.TT-3attitude change, the FDIs revealed the opinion of the experimental group students about SG and GM:Regarding the ...particularly useful in changing attitudesGM-3...a non-embarrassing learning environmentSG-1...suitable and worthwhile for the delivery of sexual health educationSG-7I was excited about the activities, competitions, leader-boards, badges, avatars, and scenariosGM-4By contrast, the control group students commented:I was bored listening to lecturesTT-1...[it was] hard to understand how the sexual health subject is important for changing my attitudeTT-7...[it is] unfriendly learningTT-6...[it was an] uncomfortable learning environment due to the sensitivity of the topic taughtTT-5...it hides potential information for changing my attitudeTT-4...questions were not encouraged or not well clarified; hence I ended up with no clues that could change the myths [that produce] negative sexual health attitudesTT-2knowledge:Experimental group students reported having had positive interactions with the SG and GM interventions and a substantial improvement in their sexual health I acquired the required knowledge for practicing healthy sexual behaviours through this learning approachGM-6I acquired potential sexual health knowledge that will help my making informed decisionsSG-6I will apply the skills and understanding that are essential and applicable for curbing unhealthy sexual behaviours.SG-4from today onwards I will not participate in risky sexual activities as I am [now] knowledgeable and will apply the knowledge acquired to make informed decisions for better sexual health outcomes and future goals.SG-4By contrast, control group students commented on their teaching method as follows:traditional teaching was less informativeTT-3technical language was used that made it difficult to understandTT-7[there was a] lack of vivid examplesTT-1the learning strategy narrowed the thinking capacity required for applying the knowledge and skills acquiredTT-4engaging them:Finally, students in the experimental learning groups (SG and GM) reported that the SG and GM components were effective in the learning activities made our minds activeGM-6I was connected to the learning processSG-4I focused on the learning activitiesSG-4the learning made me concentrate on learning all the timeGM-2Students in SG-2 and GM-7 reported that...the learning provided opportunities for hands-on activities that made it easy to learn and remember.By contrast, the TT students reported thatour learning was indirectTT-3, TT-5, TT-2the learning was passive, as no hands-on activities were providedTT-1, TT4there was little or poor interactionTT-7I lost focus during learningTT-6The study showed that the game elements embedded in SG and GM instruction catalyzed motivation and engagement during learning and that this contributed to attitude change, knowledge acquisition, and ultimately better learning performance.The study results demonstrated that sexual health education taught using SG and GM approaches works better than TT methods. The SG and GM approaches resulted in higher test scores for knowledge acquisition than the TT control group. This finding conforms with previous research which found gamified learning systems to have a significant impact on sexual health education . In our This study found that gamified learning contents were experienced as “relevant.” For instance, the students realized that there was a common connection between what they were learning and real life. These results were consistent with an earlier study , which hThe second factor accounting for the effectiveness of the gamified learning was that it changed the attitude of the students. As a similar study  reportedDuring the gamified design and development, students provided input on the type of avatars and scenarios they found appealing. Their opinions were based on their different cultural settings, their level of digital literacy, their use of state-of-the-art technology, among others. This study was grounded on the social–cultural theory known as activity theory that encourages participation of different stakeholders in the development of instructional interventions -54. MembThe third factor accounting for the efficacy of gamified learning was that it improved the knowledge acquisition among students in the experimental groups. The students indicated that gamified learning helped them to acquire sexual health information and skills that could purportedly help them engage in healthy sexual practices. Notably, the students reported that the sexual health knowledge delivered through gamified instructions were highly effective for their current and future lives and that they now felt knowledgeable and able to resist detrimental sexual health risks or factors. According to Chu et al , gamifieThe fourth and final factor that boosted students’ learning was engagement. The evaluation of the effectiveness of each of the instructional approaches was based on the students’ engagement. Studies have documented the effectiveness of gamified learning in engaging students during learning ,42,43, wThis study used a participatory design approach. Such an approach is vital in designing instructions that address the needs of the users ,43 in thmotivation and engagement that are enabled by the game elements and mechanics. The My Future Begins Today  design increases knowledge acquisition and attitude change. Students reported the learning to be more interactive through participating in the gamified learning activities.Educational gamified learning (GM and SG) has the potential to significantly increase the sexual health literacy of adolescents. The digital health gamified interventions designed in this study provide a user-friendly learning environment. The designs were influenced by a theory-driven assessment of learning. This assessment is supported by testing the learning of the users. This study treats serious digital health gamified instructions as a brain activator: it keeps students active during the learning process. The students’ participation in the learning process is catalyzed by the SG and GM methods were found to be effective and efficient in increasing motivation, improving attitudes, increasing knowledge acquisition, and encouraging engagement in the learning process. Future empirical studies may verify the efficacy of the My Future Begins Today learning platforms in improving sexual health literacy acquisition in other countries, especially in SSA, where the TT method is widely practiced and tends to limit the learning with gamified digital technologies and process ,25. This