In this study, we aim to analyze audiometric outcomes of middle ear surgeryin patients with congenital middle ear anomalies.In this single center retrospective cohort study, audiological outcomes wereextracted from patient files. Patients with a congenital middle ear anomalytreated surgically in a tertiary referral center between June 2015 andDecember 2020 were included. Pre- and postoperative short- and long-termaudiometric data (at ≥3 and ≥10 months respectively) were compared toanalyze hearing outcomes.Eighteen ears (15 patients) were treated surgically with an exploratorytympanotomy. At short term follow up statistically significant improvementsin air conduction thresholds and air-bone gaps were found. Hearing improvedin 94.4% (17/18) of operated ears. Successful outcome, defined as anair-bone gap closure to within 20 dB after surgery, was reached in 44.4%(8/18). Serviceable hearing (air conduction ≤30 dB) was reached in 55.6%(10/18). Negative outcome (any significant deterioration in hearing)occurred in 1 patient: in this ear otitis media occurred during thepostoperative course. At long term follow up, available for 50% of thecohort, hearing remained stable in 5 ears, improved in 1 ear anddeteriorated in 3, all of which underwent revision surgery. Sensorineuralhearing loss due to surgery, or other complications, were notencountered.middle ear surgery was found to be an effective treatment option to improvehearing in this cohort of patients with congenital middle ear anomalies.Surgical goals of obtained gain in air conduction thresholds and serviceablehearing levels were met by most patients without the occurrence of anyiatrogenic sensorineural hearing loss. During the embryological development the auditory ossicles are formed frombranchiogenic origin and malformations can occur at any stage of this development. Typically, conductive hearing losses of 30 to 50 dB are encountered in CMEApatients. CMEAs manifesting as isolated anomalies of the auditory ossicles areconsidered minor ear anomalies. When additional tympanic membrane or other externalauditory canal anomalies  are present, this deficit is described as amajor anomaly. The incidence of CMEAs is around 0.28 per 100,000 persons, and CMEAs occur both uni- and bilaterally. CMEAs are predominantly sporadic,yet are also described to be part of a syndromal diagnosis in more than 25% of cases.A rare cause of congenital conductive hearing loss (HL) are congenital middle earanomalies (CMEAs). They are defined as malformations of the auditory ossicles of any type.517 If ventilation tubes do notprovide sufficient improvement of hearing in case of assumed OME, or if conductivehearing loss persists after resolution of OME, further audiological assessment andimaging of the middle ear, using high resolution computed tomography (HR-CT) areindicated for patients of all ages.18 When a CMEA is suspected,especially when both ears are affected, hearing improvement is usually sought toreach functional hearing levels. In these cases conventional hearing aids, boneconduction devices, and exploratory tympanotomy (ET) are options to be considered.Surgery may be postponed until children reach the age of 10 years to minimize thedetrimental influence of postoperative otitis media on the reconstructed ossicularchain, and the risk of deaf ears due to subsequent labyrinthitis.Diagnosis of CMEAs can be significantly delayed, since conductive HL in children iscommon and often related to the high incidence of chronic otitis media with effusion(OME). Therefore, and regardless of a possible history of chronic OME in childhood,a CMEA needs to be considered in patients presenting with conductive or mixed HLlater in life. A diagnostic delay is illustrated in many studies in which CMEAs wereconfirmed surgically in adult patients of 20 to 70 years of age. In this grading system CMEAs are divided into 4 classes. According to theliterature, class 1 anomalies are the most prevalent of all CMEAs. Though more recently, it is suggested that type 3 anomalies occur mostoften.132022 In contrast to results inCMEA class 1 to 3 anomalies, surgery performed on class 4 anomalies has resulted inmixed long-term outcomes in several studies due to iatrogenic hearing loss andre-obliteration of the newly created window to the inner ear.24 Also, inseveral studies it is suggested that middle ear surgery in patients with specificsyndromes can be less effective to restore hearing than in non-syndromalpatients.16CMEAs are most widely classified according to findings during ET using the Cremersand Teunissen classification .1 In tThe decision to operate on CMEAs is made individually based on the estimated shortand long-term benefit patients achieve after surgery, and is weighted against thepossible risks during and after surgery . This to attain maximal patient and parentalinformed consent in decision making before opting for surgery.In this study we will assess hearing outcomes for a cohort of consecutively operatedpatients of varying ages with diverse anomalies of the middle ear classified asCMEAs. The aim of this study is to add to the existing literature regarding surgicaloutcomes in CMEA patients by presenting a unique cohort including all patientstreated surgically during a 5-year period without excluding adults, syndromalpatients, patients with otologic comorbidities, mixed hearing loss or a history ofchronic OME as has often been the case in previously reported cohorts. These resultswill be compared to the current literature.This is a retrospective cohort study in which patient charts were reviewed tocollect audiometric outcomes of patients with congenital middle ear anomaliessurgically treated between June 2015 and December 2020. Patients were operatedat the University Medical Center Utrecht The Netherlands, a tertiary referralcenter for middle ear disease. All surgical procedures were performed byexperienced otological surgeons .Included patients were identified from the contributing surgeon’s personaldatabases. Inclusion criteria were: (1) patients with a pre- or peroperativeCMEA diagnosis, (2) patients that received reconstructive middle earsurgery.Ethical (non-WMO) approval for this study was granted by the local review boardof the University Medical Center Utrecht, The Netherlands, local ethics number:20-792. This study was conducted in accordance with the international ethicalstandard of the Helsinki declaration (2013). as displayed in Surgical records were analyzed by one researcher (SH) to collect data aboutdemographics, medical history of the patient , type of surgical procedure performed, possible per- andpostoperative complications and audiometric outcomes. The type of CMEAs, in allcases, were classified using the Cremers classification All audiometric testing was performed in a sound treated room in themedical center’s facility by trained audiologists. Pre- and postoperativebone-conduction and air-conduction thresholds were measured at 0.5, 1, 2, and4 kHz. Pure tone averages (PTAs) were calculated averaging these 4 values.Air-bone gaps (ABGs) were calculated using PTAs of BC and AC thresholds. When BCimproved postoperatively (overclosure), the preoperative ABGs were corrected byusing the postoperative BC. Successful hearing outcome was defined as closure ofthe ABG ≤ 20 dB. Serviceable hearing was defined as AC threshold ≤30 dB. A negative hearing outcome was defined by no change or any worsening inAC threshold PTAs or a worsening in BC PTAs ≥ 10 dB if the deterioration was adirect cause of the performed middle ear surgery. Hearing (AC) was consideredstable if AC remained within 5 dB of the postoperative AC value used tocalculate results. Individual short term audiometric results were visualizedusing Amsterdam Hearing Evaluation Plots (AHEPs).Hearing outcomes were collected out of the audiometric evaluations within4 months before surgery and at ≥3 months or longer (<10 months) after surgeryto assess short-term postoperative outcomes. Hearing outcomes from theaudiometric evaluation closest to 3 months postoperatively were used. To assesslong term outcome, hearing outcomes measured at 10 months or longerpostoperatively were collected. Hearing outcomes from the latest availableaudiometric evaluation >10 months postoperatively were used. If revisionsurgery was performed, the last available audiometry before revision surgery wasused. Collection of audiometric data was done in accordance to the committee ofhearing and equilibrium guidelines.P-value of <0.05 was considered significant.SPSS (IBM SPSS statistics 25.0.0.2) was used to perform all statistical analyses.Means, standard deviations and ranges were determined of pre- and postoperativeaudiometric results, that is, AC- and BC-threshold PTAs and ABGs. Using theWilcoxon signed rank test preoperative audiometric results were compared topostoperative audiometric results for short and long term follow-up. AFifteen patients (18 ears) underwent a total of 21 surgical procedures  to restore middle ear function impaired by CMEAs.All patients had a history of hearing loss since early childhood. Other possiblecauses of hearing loss for example, ossicular fixation due to multiple episodes ofotitis media or otosclerosis were excluded by combining anamnestic data withreferrals and findings during exploratory tympanotomy.Each ear and its specifications are displayed per patient in ®, Dresden), was performed. For class 2 anomalies acombination of a stapedotomy procedure and the reconstructions conducted inclass 3 anomalies was performed. During stapedotomy the stapes suprastructurewas removed, the footplate was perforated (using a 1W KTP laser) and a TeflonCausse piston  was placed.All surgical procedures were performed under general anesthesia, using atrans-canal endaural (16 procedures) or retro-aural approach (5 procedures) toreach the middle ear. The used technique for ossicular chain reconstructiondiffered depending on the found anatomical anomalies of the ossicular chain andconsequential classifications. For class 1 anomalies a stapedectomy procedurewas performed : the staP < 0.001), comparing thepreoperative and short term postoperative measurements. In 17 out of 18 ears(94.4%) improvement of AC was obtained comparing preoperative measurements toshort term postoperative measurements. Successful outcome (ABG ≤ 20 dB) wasreached in 8 out of 18 (44.4%) ears  and 5 out of 10 adults (50.0%). Serviceable hearing levels (AC ≤ 30 dB)were reached in 10 out of 18 ears (55.6%), in 75% (6/8) of children and in 40%(4/10) of adults. No postoperative decline in bone conduction hearing levels were seen in this study. Negative outcome was seenin one (5.6%) out of 18 ears. In this patient  a postoperative acute otitis media occurred 3 weeks afterossicular chain reconstruction (OCR) surgery. At hearing evaluation 6 weeksafter surgery an AC PTA of 76.3 dB, BC of 36.3 dB and ABG of 40.0 dB were foundcompared to preoperative PTAs AC 63.8 dB, BC 40.0 dB and ABG23.8 dB. Thesehearing levels remained stable during the 5 years of FU after surgery. It waspatient’s and doctor’s shared decision that revision surgery should not beperformed as the low probability of relevant hearing improvement did notoutweigh the risk of a postoperative deaf ear.Short-term postoperative hearing outcomes were available for all 18 ears and aredisplayed in Long term (FU ≥ 10 months) postoperative hearing outcome was available for 9 outof 18 ears (50.0%). The mean duration of long-term follow up was 23.6 months(10.8-58.9 months). Mean postoperative audiometric values were: BC 13.8 dB, AC44.3 dB, and ABG 30.6 dB . Postoperative hearing outcomes remained stable in 5 out of 8cases  and improved in 1 case (ear 17). Hearingdeclined in the other 3 cases , which were all class 3 CMEAs,all of which underwent revision surgery.®, titanium prosthesis) (length 2.5 mm) was placed. Thisresulted in a subjective improved hearing directly postoperatively, however6 days after surgery hearing loss without vertigo or instability occurred inthis ear  which was interpreted as sudden deafness andtreated accordingly with 7 days of prednisone. HR-CT and MRI did not showabnormalities of the middle ear and position of the placed prosthesis. Hearingremained stable in the 2 months follow up after this event (BC 66.3 dB and AC88.8 dB) and no further explanation for the HL was found.Postoperative hearing in ear 3, after PORP placement to correct a missing long process of the incus, improved from BC31.3 dB, AC 81.3 dB, and ABG 50.0 dB preoperatively to BC 36.3 dB, AC 56.3 dB,and ABG 20.0 dB postoperatively. Recurrence of conductive HL occurred  19 months after surgery. Preoperative HR-CTbefore revision surgery demonstrated a correct position of the used ossicularreconstruction prosthesis. During revision ET the previously placed PORPappeared too short (length 2.0 mm) and a new, longer PORP  was positioned on the mobilestapes suprastructure. Hearing and audiometric PTAs improved  and remained stable during follow up (5 months).After initial surgery in ear 9, in which the fixation of the anterior mallearligament was removed, hearing improved according to the patient and thengradually declined in the 8-week period following with audiometrical outcomes ofBC 8.8 dB, AC 27.5 dB, and ABG 18.8 dB compared to BC 2.5 dB, AC 35 dB, and ABG32.5 dB preoperatively. Revision ET revealed recurrent fixation of the anteriormallear ligament. The incus and fixated malleus head were removed, and a PORP was placed resulting in improvedhearing .Surgery in ear 15 (hearing loss by 22.q.11.ds), in which the anterior fixation ofthe mallear manubrium was removed, resulted in little hearing improvement. Audiometry 1 year post surgery showeddeterioration in hearing levels . Revisionsurgery was performed in which the long leg of the incus was removed and a PORPof ears had ABGs ≤ 20 dB after surgery (and revision surgery) compared to zero outof 18 before surgery. The results of this study were compared to previouslypublished patient series that reported hearing outcomes in larger patient cohorts(n ≥ 10) and followed the AAO-HNS committee guidelines for reporting hearingoutcomes.29 which iscomparable to our experience . Iatrogenic sensorineural hearing loss(SNHL), although rare, is reported to occur most frequently in CMEA class 1cases (0% to 4%)29 compared to the other classes of CMEAs. This can beexplained, considering the increased risk of an infectious event after openingthe inner ear during stapedectomy procedures. No surgery related SNHL occurredin our cohort.In the literature good results of surgery on class 1 ears have been reported withmean postoperative ABGs of 11 to 14 dB and success rates of 74%,1029 The presented cohortincluded 2 class 2 ears in which a gain in AC was achieved of 17.5 dB andsuccess rate of 0% (mean postoperative AC 40.0 dB and ABG 26.9 dB). Ear 2 had anotologic history of multiple episodes of otitis which is likely to have limitedfinal hearing outcome. During surgery on this ear atelectasis of the tympanicmembrane and many adhesions of the middle ear were found.Literature in which hearing outcome on class 2 patients is reported is limited.In the few reports that published outcome in class 2 patients an AC gain of 18to 20 dB has been found, with higher postoperative ABGs (13-20 dB) and lowersuccess rates (67% to 70%) than in class 1 and class 3 patients.31 The mean postoperativeABG and gain in AC achieved in our cohort are rather similar to published caseseries, but success rates were lower (44.4% in our cohort compared to a rangefrom 56.1% to 75.0% in the literature). These lower success rates might beexplained by the significant amount of ears (7/14) with an otologic history ofchronic OME. Additionally, 3 of these ears  presented with highAC PTA’s and large ABGs which decreases the probability of closing the ABG towithin 20 dB. Furthermore, the class 3 group included all 3 syndromic ears ofwhich 2 (ears 11 and 15) did not acquire a postoperative ABG within 20 dB.Most ears in this study (14/18) were classified as class 3. Results on class 3ears presented in the literature are summarized in 32 or excluding those patients with mixed hearing loss,inner ear anomalies, otologic comorbidities and a history of recurrent otitismedia.1333 Second, long term hearingoutcome of 10 months or longer after surgery was only available for half thecohort (9 out of 18 patients).In this study we analyzed the results of reconstructive surgery on hearing forall surgically treated CMEA patients in our medical center. The detaileddescription and analysis of included cases provides knowledge on the expectedoutcome of reconstructive surgery of the ossicular chain in these patients.Though, several limitations needs to be considered. First, we included a cohortof children and adults who were surgically treated for CMEAs. Thereby, outcomescould differ to previously published hearing outcomes in cohorts only includingchildren, Exclusive non-surgical hearing improvement might be considered inunilateral patients in which postoperative hearing gain is less likely, forexample, patients with extensive otologic histories, with class 4 anomalies,with specific syndromal diagnoses, and in patients with inner ear deformities. Also, the importance of preoperative radiological imaging and carefulinspection of the ossicular chain during ET for additionalfixations/malformations needs to be mentioned as these factors could hindersuccessful outcome.In this retrospective chart review, we demonstrated that surgery is a clinicallyrelevant treatment option for CMEAs to reach AC thresholds below 30 dB toachieve functional hearing levels. Furthermore, even if AC PTAs remain>30 dB, improved hearing can be of importance to the patient by facilitatingadditional hearing amplification by hearing aids.Surgery proved to be an effective treatment option to restore hearing for CMEAs.Surgical goals of obtained gain in AC thresholds and reached serviceable hearinglevels were met by most patients without any iatrogenic sensorineural hearing lossesor other inner ear problems.


Extracellular vesicles (EVs) from peritoneal dialysis effluent (PDE), containing molecules such as proteins and microRNAs (miRNAs), may be potential biological markers to monitor peritoneal function or injury. Peritoneal inflammation is an important determinant of peritoneal solute transport rate (PSTR). Thus, the aim of this study is to determine whether the specific proteins capable of evaluating the PSTR could be found in PDE-EVs, and explore the underlying mechanism for the association between PSTR and peritoneal inflammation.Sixty patients undergoing peritoneal dialysis (PD) were divided into two groups: high/high average transport (H/A) group (PET >0.65) and low/low average transport (L/A) group (PET <0.65). EVs derived from PDE (PDE-EVs) were isolated by ultracentrifugation. Proteomic analysis was performed to explore the differentially expressed proteins and identify the potential biomarkers in PDE-EVs from the two groups, and we focused on glycoprotein 96 (GP96) as it could be involved in the inflammatory process. The expression of GP96 in PDE-EVs and inflammatory cytokines was quantified by real-time PCR and enzyme-linked immunosorbent assay. The infiltration of macrophages and neutrophils into the peritoneum was detected using immunohistochemistry in a PD rat model.The expression of PDE-EVs-GP96 was significantly higher in the H/A group, and was positively correlated with the PSTR and the level of the inflammatory factor interleukin (IL)-6. GP96-enriched EVs enhanced the secretion of proinflammatory cytokines IL-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-8 in macrophages, which was reversed by a pharmacological GP96-specific inhibitor (PU-WS13). The GP96 inhibitor also reduced local peritoneal inflammation by decreasing the infiltration of inflammatory cells and levels of proinflammatory cytokines (IL-6 and TNF-α) and chemokines  in a PD rat model.PDE-EVs-GP96 is a new promising tool to evaluate the status of peritoneal inflammation and PSTR, and the mechanism may be related to affecting the inflammatory properties of macrophages. Intraperitoneal inflammation may be essential for peritoneal dysfunction in PD patients, and the PSTR reflects local inflammation within the peritoneal cavity  have vastly different capabilities for removing or transporting solutes and water embranes , 2. Highembranes –5. The tl cavity . Severall cavity –12. The l cavity . Furtherl cavity , 12.Extracellular vesicles (EVs), including exosomes, ectosomes, and apoptotic bodies, are nano- or micron-sized vesicles, that play an important role in intercellular communication, as they transfer biologically active molecules, including proteins, lipids, and nucleic acids, to target cells , 14. RecSubstantial research has been undertaken to clarify the role of EVs in inflammation and inflammatory diseases. Proteomics is used to study EVs in inflammatory diseases, mainly to monitor changes in the expression or modification of proteins in normal versus pathologic states, which enables the identification of novel biomarkers and clarification of the underlying mechanisms in inflammatory diseases , 21. Altin vitro, and then used a GP96 inhibitor to observe its role in inflammation. We also observed the effect of the GP96 inhibitor on peritoneal inflammatory cell infiltration and inflammatory cytokine secretion in a PD rat model. Collectively, our findings highlight PDE-EV-GP96 as a promising molecular marker to evaluate peritoneal transport characteristics and even a promising therapeutic target in peritoneal inflammation in PD patients.In this study, we used data-independent acquisition (DIA) technology for quantitative proteomics analysis, to explore the differentially expressed components in PDE-EVs from PD patients with different PSTRs, and to identify key proteins that were enriched in pathways of the immune inflammatory response. We hypothesized that GP96 may be a key protein associated with PSTR or peritoneal inflammation and verified this in clinical samples. Furthermore, we selected GP96-enriched EVs to stimulate macrophages We conducted a single-center study of 60 PD patients followed up at the Peritoneal Dialysis Center Department of Nephrology of Shanghai Ninth People’s Hospital. Inclusion criteria were as follows: (1) age 25–75 years, (2) had undergone regular PD therapy (defined as 3– 4 exchanges/day), (3) dialysis for >24 months, (4) serum albumin level ≥35 g/L, and (5) had been clinically stable for at least 3 months before the assessment. Patients with acute coronary syndromes, atrial fibrillation, severe valvular heart disease, cerebrovascular disorders, chronic lung disease, chronic liver diseases, infectious or non-infectious inflammatory diseases, malignancies, mental diseases, abuse of drugs or alcohol, pregnancy, and renal transplantation were excluded from the study.Cr, which were denoted as “high/high average ” or “low/low average .” The PSTR was considered to be stable if no changes in the PET results were detected three times in succession. We enrolled 60 patients with stable PSTRs and nocturnal dwell prescriptions in our study. Overnight peritoneal dialysate samples were collected after exchanging 2 L of PD solution  for 10 h. The dialysate was then drained and the remaining amount was measured.Peritoneal transport characteristics were assessed using the criteria defined by Twardowski et al. . PatientClinical data including age, sex, history of disease, use of medication, body mass index, dialysis duration, diabetes, hypertension, and 24-h UF volume were recorded. The causes of end-stage renal disease are mostly diagnosed based on clinical features.g) at 4°C to remove cellular debris. After centrifugation for 30 min (18000 ×g) at 4°C, the supernatants were filtered through a 0.22-μm filter unit to remove larger particles, and then centrifuged for 2 h (120000 ×g) at 4°C using an ultracentrifuge  to obtain EVs. The remaining precipitate was washed with phosphate-buffered saline (PBS) and centrifuged at 120000 ×g for 2 h. The final pellets were then redissolved in 200 μL of PBS and stored at −80°C for further experiments.The samples (500 mL of PDE per sample) were concentrated using a stirring UF cell using 10 kDa NMWL Ultracel regenerated cellulose membranes  to obtain 40 mL of concentrated PDE per sample. The concentrate was centrifuged for 15 min  was performed to identify the morphology of the obtained PDE-EVs. The PDE-EVs suspension was fixed in buffered 4% paraformaldehyde for 60 min at 4°C. Then, 10 μL of each sample was added to a formvar-coated copper grid and incubated for 10 min at room temperature. After washing with PBS, the grid was stained with 3% aqueous phosphotungstic acid for 2 min and finally evaluated using TEM at an acceleration voltage of 100 kV.The size and concentration of PDE-EVs were measured by NTA , which is a combination of light scattering and Brownian motion technology. Each EV sample was diluted in pre-filtered PBS to an optimal concentration and then injected into the measurement room. The device performs measurements at 11 different locations in the measurement room and gives an average value. Each vesicle sample was measured three times to obtain the average value. Data were analyzed using ZetaView version 8.04.02.Single-pot, solid-phase-enhanced sample-preparation (SP3) technology was used to digest 10 H/A-EV and 10 L/A-EV samples. The detailed operational procedures have been described in our previous study . Briefly6, respectively.The enriched peptides of all samples were analyzed by an in-house packed capillary column  at 250 nL/min, and the gradient was as follows: 2 min, 5-8% B (0.1% (v/v) FA in 84% ACN); 80 min, 8-23% B; 26 min, 23-45% B; 2 min, 45-100% B; 10 min, 100% B. DIA (Data-dependent acquisition) technology was applied for the acquisition using a mass spectrometer (Q Exactive HFX) coupled with nanoLC1200 system  was applied to associate Gene Ontology (GO) terms with the differentially expressed proteins, and then functional enrichment analysis was performed using the online platform Database for Annotation, Visualization, and Annotated Discovery. The GO enrichment analyses consisted of three categories: biological process (BP), molecular function (MF), and cellular component (CC). All functional annotations were obtained from the UniProt database.Spectronaut 12.0 Pulsar (Biognosys) was used to analyze all raw files to generate an assay library for DIA analysis using default settings. The detailed information has been described in our previous study . PotentiHuman monocytic THP-1 cells (THP-1) were obtained from ATCC  and cultured in RPMI 1640 medium  containing 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). THP-1 monocytes were differentiated into macrophages by supplementing with 150 nM phorbol 12-myristate 13-acetate  over 24 h and starved in fresh RPMI 1640 medium overnight.The effect of PDE-EVs on macrophage function was assessed to demonstrate the biological activity of GP96 in PDE-derived EVs. PDE-EVs were labeled with a PKH67 Green Fluorescent Cell Linker Mini Kit  according to the manufacturer’s protocol. Macrophages were incubated with PKH67 labeled EVs for 24 h and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Then, the labeled EVs were captured by living macrophages. The localization of EVs (green) and nuclei (blue) in the stained macrophages was observed using a fluorescence microscope to verify the uptake and internalization of the PDE-derived EVs.The concentration of PDE-EV GP96 in the H/A and L/A groups was also quantified by an enzyme-linked immunosorbent assay (ELISA). Macrophages were incubated with H/A-PDE EVs , L/A-PDE EVs  (n=4), or H/A-PDE EVs combined with 0.5 μM PU-WS13 for 24 h, and changes in cytokine levels were measured by polymerase chain reactions (PCR) and western blotting. PBS was added to the control group.4 cells/well) and separately incubated with different PU-WS13 concentrations  for 24 h. Then, 10 ul of Cell Counting Kit-8 (CCK-8) reagents  were added to each well for 2 h at 37°C. The absorbance was measured using a microplate reader  at 450 nm (n = 6 for each group).The macrophages were seeded into 96-well plates . Tissues from the peritoneum were collected and placed in 10% paraformaldehyde for immunohistochemical analysis or plunged into liquid nitrogen for PCR and western blotting.All rats received appropriate care in accordance with the animal protocols approved by the Animal Care and Use Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine. All experiments were performed in accordance with the recommended guidelines. Thirty male Sprague-Dawley (SD) rats, weighing between 120 and 160 g, were obtained from the Chinese Academy of Sciences . All rats were housed with a 12 h light/dark cycle and had free access to food or water throughout the experiment. Animals were randomly divided into three groups: CKD rats (n=10), PD rats (n=10), and 17-AGG rats (n=10). All rats underwent a subtotal nephrectomy. Four weeks after subtotal nephrectomy, the rats underwent surgery to implant a peritoneal catheter connected to a subcutaneous mini access port for the acute model of exposure to 4.25% Dianeal  or saline . The CKD group was instilled with saline. The PD group was instilled with peritoneal dialysis fluid (PDF). For 17-AGG experiments, PDF-instilled rats were treated with 17-AGG  every 3 days 2O2 for 5 min at room temperature. The sections were incubated with primary antibodies specific for CD68  and MPO  overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated anti-mouse secondary antibody for 60 min. Antibody binding was visualized using diaminobenzidine.Peritoneum samples were collected and fixed in buffered 4% paraformaldehyde at room temperature, and then incubated for 1 h at room temperature in PBS containing 10% goat serum, 0.3 M glycine, 1% bovine serum albumin, and 0.1% Triton, which could permeabilize the cells and block non-specific protein-protein interactions. Then, peritoneum sections were treated with 3% HThe number of macrophages or neutrophils was calculated by measuring the positive area of representative images  and analyzed using ImageJ .Protein was extracted from isolated EVs with RIPA  and All-In-One buffers . A BCA Kit was used for protein quantification (Solarbio). Western blotting was performed as described in our previous study . PrimaryTotal RNA was extracted from the macrophages or peritoneum samples using TRIzol (Sigma), and then reverse transcribed into cDNA using Prime Script RT Master Mix . The cDNAs were subsequently used for gene expression by quantitative real-time PCR with the TB Green Premix Ex Taq kit . The PCR program was as follows: denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and annealing at 60°C for 34 s. The primers used are listed in g. Then, the supernatant was collected and used for ELISA analysis of inflammatory cytokines. The cytokines, including TNF-α, IL-6, IL-1β, and IL-8, were examined using an ELISA kit  according to the manufacturer’s recommendations. The minimum threshold concentrations of the TNF-α, IL-6, IL-1β, and IL-8 proteins detected by each ELISA kit were 15.6, 7.8, 2.0, and 15.6 pg/mL, respectively. Briefly, 100 µL of each sample or standard was added to the previously capture antibody-coated plate and incubated for 90 min at 37°C. After removing the liquid,100 µL of Biotinylated Detection Ab was added and incubated for 60 min at 37°C. After three washes, 100 µL of HRP conjugate was added to each well and incubated for 30 min at 37°C. After another three washes, 90 µL of substrate reagent was added and incubated for 15 min at 37°C. Then, 50 µL of stop solution was added and the absorbance was measured using a microplate reader .PDE-EVs were extracted from 500 mL of PDE as described above. The concentration of GP96 in PDE-EVs was measured using commercial ELISA kits . The minimum detected threshold concentration of GP96 protein was 0.31 ng/mL (n=20 for each group). PDE or cell culture suspension was centrifuged for 15 min at 2500 ×t-test for two groups, and one-way analysis of variance (ANOVA) for multiple groups. Spearman correlation was used for correlation analysis. All data analyses and graph generation were performed using IBM SPSS Statistics 23  and GraphPad Prism 8.0.1 . A p-value <0.05 was regarded as statistically significant. The enriched GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were visualized using R-Studio software .Results are presented as the mean ± standard deviation (SD). Statistical differences were determined using an unpaired Student’s EVs were isolated from concentrated overnight PDE samples by differential centrifugation. To identify the PDE-EVs, we visualized EVs with the typical rounded, cup-shaped bilayer morphology through TEM  and those of 33 were downregulated  in the H/A group compared with the L/A group. Cluster analysis was performed for the differentially expressed proteins , activate relative inflammatory signaling pathways, and ultimately release a large number of pro-inflammatory cytokines . In total, 50 µg of total protein from each sample was added to each well for western blotting. A band at 100 kDa corresponding to GP96 was detected in all samples, and this band had variable intensity between samples . PDE-EVs were extracted from 500 mL of overnight PDE per sample. The clinical data of these patients are summarized in Cr, UF volume, and inflammatory indicators. The expression level of GP96 in PDE-EVs was positively correlated with 4 h D/PCr , whereas the correlation between 4 h UF or 24 h UF with GP96 in PDE-EVs was not significant , PDE-EVs from the H/A group (H/A-EVs), and PDE-EVs from the L/A group (L/A-EVs) were used to compare the immunomodulatory effects of PDE-EVs from both groups. To attain PDE-EVs containing >100 ng/mL GP96 to activate macrophages, based on previous studies , 34, EVsp <0.01; p >0.05; Next, we assessed whether macrophages treated with a GP96-specific inhibitor could reduce proinflammatory response. Macrophages that were stimulated with H/A PDE-EVs were treated with 0.5 µM PU-WS13. PU-WS13-treated macrophages experienced a significant decline in the levels of proinflammatory molecules IL-6, IL-1β, TNF-α, and IL-8 induced by H/A PDE-EVs  infiltration was evaluated by immunohistochemistry (IHC). Daily instillation of PDF caused a marked increase in macrophage and neutrophil infiltration in the peritoneum of the PD group  was applied to a rat model of peritoneal dialysis. 17-AGG is not a pharmacological GP96-specific inhibitor, but it binds to GP96 in the ATP-binding site, thereby inhibiting its ATPase activity . To asseIn the present study, we found that the expression levels of GP96 in EVs from PDE were significantly higher in patients with high PSTRs and positively correlated with the PSTR and dialysate IL-6 concentration. We verified that GP96-enriched EVs stimulated macrophages to secrete more proinflammatory cytokines, and that the GP96 inhibitor attenuated local inflammatory cell infiltration and proinflammatory gene expression in the peritoneum of a rat peritoneal dialysis model.MicroRNAs or proteins in EVs are promising biomarkers for several diseases and pathologic states , 36, andProteins related to EVs endocytosis and the immune response are enriched in EVs from high transporters. Some of these proteins, such as heat shock proteins (HSPs) and S100 proteins, are involved in exosome-mediated immunoregulation or inflammation , 39. SevTo identify the role of proteins in PDE-EVs during peritoneal inflammation, we screened the proteins that were differentially expressed between the groups. We focused on GP96, which is upregulated in EVs derived from the PDE of PD patients. GP96, encoded by the HSP90B1, is an endoplasmic reticulum (ER)-associated chaperone of HSP90. Additionally, GP96 is constitutively expressed in virtually every cell type, and its expression is upregulated by cytosolic and ER stress, which is caused by several factors, including the accumulation of misfolded proteins, oxidative stress, and acidosis , some ofGP96 is an obligate chaperone for TLRs and multiple integrins, and plays a critical role in regulating both innate and adaptive immunity , 41. In in vivo situation than EVs from cell culture . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Care and Use Committee of the School of Medicine, Shanghai Jiao Tong University (HKDL [2018] 38). Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.FD and YL designed the research. JF and YT conducted the research and analyzed the results. JF wrote the paper. OJ and SW conducted the real-time PCR experiments and analyses. ZC, AS, and PL performed the western blotting measurements and analyses. YZ, HZ, and HR conducted the proteomic analysis. All the authors read and approved the final manuscript.This work was sponsored by the Shanghai Health Commission Clinical research project (202140430), the National Natural Science Foundation of China (8217030178), Research Program of 9th People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYLJ2018011) and Fundamental Research Program Funding of 9th People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (JYZZ058).YZ, HZ, and HR were employed by Shanghai Applied Protein Technology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Upon hearing someone’s speech, a listener can access information such as the speaker’s age, gender identity, socioeconomic status, and their linguistic background. However, an open question is whether living in different locales modulates how listeners use these factors to assess speakers’ speech. Here, an audio-visual test was used to measure whether listeners’ accentedness judgments and intelligibility  can be modulated depending on racial information in faces that they see. American, British, and Indian English were used as three different English varieties of speech. These speech samples were presented with either a white female face or a South Asian female face. Two experiments were completed in two locales: Gainesville, Florida (USA) and Montreal, Quebec (Canada). Overall, Montreal listeners were more accurate in their transcription of sentences  compared to Gainesville listeners. Moreover, Gainesville listeners’ ability to transcribe the same spoken sentences decreased for all varieties when listening to speech paired with South Asian faces. However, seeing a white or a South Asian face did not impact speech intelligibility for the same spoken sentences for Montreal listeners. Finally, listeners’ accentedness judgments increased for American English and Indian English when the visual information changed from a white face to a South Asian face in Gainesville, but not in Montreal. These findings suggest that visual cues for race impact speech perception to a greater degree in locales with greater ecological diversity. Babel and Russell  found thYi and colleagues  merged tAs Babel and Mellesmoen  assertedThis study investigates whether living in differing multilingual locales modulates speech perception and accentedness judgments towards three English varieties . These three varieties were chosen for several key reasons. Historically, these three varieties share the same linguistic past. Both American and Indian English emerged from British English, though one emerged as a result of British settlement  while the other as a result of British colonialism  Kachru, . Often, Thus, to investigate whether living in multilingual, multicultural locales modulates speech perception and accentedness judgments towards racialized varieties, we conducted two experiments: one in Gainesville, Florida (USA), and one in Montreal, Quebec (Canada). These multilingual locations offer a unique way to test whether variability in language exposure  modulates foreign accent judgments. Gainesville is a small college town in Florida hosting primarily English–Spanish bilinguals. Although there is a substantial amount of Spanish spoken in Florida, there are negative attitudes associated both with speaking Spanish , Montreal is an urban, multilingual city with a large English-French bilingual population  and Gainesville (87.4) did not statistically differ in their English proficiency. In Montreal, 7 out of 25 participants indicated that they were not born in Montreal. However, all 7 participants indicated that they had lived in Montreal for at least 1 year or more . In Gainesville, 5 participants indicated that they were born outside of Florida. These participants also indicated that they spent at least 1 year in Gainesville . Since participants live in different locales, we quantified the linguistic diversity that they encounter every day through language entropy  self-identified their speech as American English. The objective LexTale English proficiency test scores had a mean score of 86. All judges were exposed to Spanish to some degree. Additional language background questions yielded that none of the judges had extensive exposure to either British or Indian English. However, all judges indicated more familiarity  with British English compared to Indian English. During the norming task, participants did not see any visual information on the screen. They were asked to transcribe what they were listening to . For the intelligibility task, participants were first shown an image on the computer screen. About 250 ms after the onset of the image, the auditory stimulus played. The image remained on the screen throughout the duration of time that participants were typing their sentences. This manipulation was done to prime participants with the socio-indexical information of the speaker . Participants were asked to first listen to the short sentences and were then asked to start typing the sentences. Participants were instructed to ignore any punctuation and capitalization and were explicitly told to type as quickly and as accurately as possible , and each participant completed only one of the four lists.Testing was completed in a quiet room. Participants were recruited through the SONA systems in both testing universities. The experiment took 1.5 h to complete, and participants received class credits upon their completion.tels instead of tells or bok instead of book). Accuracy proportions were then calculated for each sentence (n = 120) and for each participant. To investigate accuracy proportions as a function of our independent variables—face, English variety, sentence predictability, and location—we constructed a linear mixed-effects model using the lme4 package  . No such difference was observed between American and British English . Results also yielded significant differences between two locales such that Montreal participants were overall more accurate in their transcriptions compared to Gainesville participants . These main effects were further qualified by multiple interactions.Results showed that sentences paired with white faces were transcribed more accurately than those paired with South Asian faces . Indian English paired with South Asian faces was transcribed less accurately compared to white faces . Moreover, British English paired with South Asian faces was transcribed less accurately compared to white faces . The third interaction was between Face and Location  such that speech paired with South Asian faces was transcribed less accurately compared to white faces in Gainesville , and speech paired with South Asian faces was transcribed less accurately in Gainesville compared to Montreal . The third interaction was between Variety and Location . This interaction was driven by a more accurate transcription of American and British English compared to Indian English in Gainesville  and less accurate transcriptions of Indian English in Gainesville than in Montreal .First, there was an interaction between Face and English Variety . This interaction revealed that Indian English presented with South Asian faces was transcribed less accurately in Gainesville than in Montreal  . Moreover, British English was judged as more accented compared to American English , and Indian English was judged as more accented than both American and British English combined . These main effects were also further qualified by multiple interactions.Results showed that listeners judged white faces as less accented compared to South Asian faces  and between Face (white) and Variety type (Indian vs. American + British) . These interactions suggest that for both American and British English, whenever they were paired with white faces, these recordings were judged as less accented compared to when paired with South Asian faces. This effect was not observed for Indian English (p > 0.05).There was an interaction between Face (white) and Variety type (British vs. American) , while no such difference was observed for South Asian faces (p > 0.05). Location also interacted with Variety type  such that British English was judged as less accented in Gainesville compared to Montreal . No such difference was observed for American or Indian English.There was another interaction between Face (white) and Location  which shows that white faces were judged as more accented in Montreal than in Gainesville . This interaction was driven by higher accentedness judgments towards American English paired South Asian faces in Gainesville when compared to Montreal participants  as well as British English recordings being judged as more accented in Montreal compared to Gainesville when paired with both white and South Asian faces   when presented with white and South Asian faces. To test this, we conducted two experiments in two different locales, Gainesville and Montreal, that differed in terms of their multilingualism and multiculturalism. Overall, our findings showed that living in a locale where multilingualism is not promoted modulated speech perception, particularly when it was paired with South Asian faces. We discuss these findings in detail below.First, we found locale differences such that Montreal participants were overall more accurate in their transcriptions compared to Gainesville participants. This difference could arise from the linguistic uncertainty that Montreal and Gainesville participants have in their everyday life. A recent framework proposed by Gullifer and Titone  argues tIn terms of accentedness judgments, Montreal participants did not judge speech paired with South Asian faces as more accented compared to white faces within the same variety. For instance, there was no difference between white and South Asian faces for American, British, or Indian English. However, in Gainesville, both American and British English were judged as more accented when paired with South Asian faces. These differential mechanisms that are engaged for accentedness judgments in Gainesville and Montreal suggest that the social meaning of observable race has different values in these two locales. For instance, race is a less reliable cue in Montreal compared to Florida as it was seen that Montreal participants primarily use speech variables to assess one’s accentedness level, while Gainesville participants use both face and accents to make their judgments  associate Indian English with out-group members of the English-speaking community Kachru, . HoweverThe color of a lemon is yellow) and half low-predictable sentences . For the intelligibility task, it would be ideal to have all low-predictable sentences. Importantly, we normed all recordings without any visual information to have at least 85% intelligibility. This biases speech recordings to be highly intelligible. However, everyday interactions do also contain unintelligible speech. Findings in future studies might differ depending on the norming process. Nonetheless, these experimental contradictions should be interpreted as the need for speech perception research that is geared towards understanding social information processing .We acknowledge several limitations of our design. We chose the sentences as they have been widely used in previous speech perception research. However, the sentence list consists of half high-predictable (i.e., Our results here reinforce the role of race in speech perception. Further, they speak to how multilingual environments, race, and speech perception are intertwined. More studies are needed to understand the connection between foreign accents and race and how listeners form their associations towards multilingual groups and the ways in which they converge or diverge with multilingual speakers (Walker & Campbell-Kibler,


COVID-19 has brought an unprecedented challenge to healthcare services. The authors’ COVID-adapted pathway for suspected bowel cancer combines two quantitative faecal immunochemical tests (qFITs) with a standard CT scan with oral preparation (CT mini-prep). The aim of this study was to estimate the degree of risk mitigation and residual risk of undiagnosed colorectal cancer.Decision-tree models were developed using a combination of data from the COVID-adapted pathway (April–May 2020), a local audit of qFIT for symptomatic patients performed since 2018, relevant data  obtained from literature and a local cancer data set, and expert opinion for any missing data. The considered diagnostic scenarios included: single qFIT; two qFITs; single qFIT and CT mini-prep; two qFITs and CT mini-prep (enriched pathway). These were compared to the standard diagnostic pathway ).The COVID-adapted pathway included 422 patients, whereas the audit of qFIT included more than 5000 patients. The risk of missing a colorectal cancer, if present, was estimated as high as 20.2 per cent with use of a single qFIT as a triage test. Using both a second qFIT and a CT mini-prep as add-on tests reduced the risk of missed cancer to 6.49 per cent. The trade-off was an increased rate of colonoscopy or CTVC, from 287 for a single qFIT to 418 for the double qFIT and CT mini-prep combination, per 1000 patients.Triage using qFIT alone could lead to a high rate of missed cancers. This may be reduced using CT mini-prep as an add-on test for triage to colonoscopy or CTVC. Triage of symptomatic patients referred to a colorectal service using a single quantitative faecal immunochemical test alone leads to an unacceptably high rate of missed colorectal cancers based on modelling. This may be effectively reduced by using standard CT with oral preparation as an add-on test for triage to colonoscopy or CT virtual colonoscopy. COVID-19 has brought unprecedented challenges to health and social services. The standard diagnostic pathway for suspected colorectal cancer (CRC) was severely curtailed at the onset of the pandemic with the majority of hospitals missing cancer targets,Many institutions have proposed alternative strategies to circumvent this situationWhilst new measures are required to rationalize diagnostic resources and allocate tests to those who are most likely to have pathology, the sensitivity of the qFIT remains low compared with the established diagnostic tools in symptomatic patients. False-negative results, using qFIT as a single diagnostic test at a threshold of 10 µg haemoglobin/g faeces (µg/g), result in missed cancersThe Western General Hospital and University of Edinburgh have been operating a COVID-adapted diagnostic pathway, which combines multiple qFITs and a standard CT scan with oral preparation without rectal insufflation (CT mini-prep) to rule out gross colorectal pathologyThis paper aimed to develop models to calculate the degree of risk mitigation and estimate residual risk of CRC of the COVID-adapted pathway, considering selected alternative patterns of diagnostic pathways, and comparing them against the pre-COVID conventional diagnostic pathway.Decision-tree models were developed using a combination of data from the COVID-adapted pathway and a large local audit of the use of qFIT in symptomatic patients, literature review of diagnostic test accuracy and expert opinion.The local audit of qFIT for symptomatic patients has been running since 2018 at NHS Lothian. qFIT has been used as agnostic of primary care decisionand was not utilized as a gatekeeping tool to access secondary care. There are 85 data items that have been collected in the local audit data set, including demographics, presenting history and symptoms, co-morbidities, blood results at the time of referrals and the outcome of standard diagnostic investigations . In FebrThe specific data derived from the COVID-adapted pathway were the sensitivity of CT mini-prep to diagnose grossly abnormal pathology, mainly CRC. CRC detection rate at the unit was calculated from a data set compiled by South East Scotland Cancer Network and the number of referrals to the department in preceding years. As most of the data about qFIT in the literature were based on asymptomatic screening populationsThe details of the COVID-adapted pathway have been published previouslyCT scan and qFIT if deemed 'high risk'Only qFIT when the referral was deemed 'low risk' or did not meet the urgent referral criteriaFast-track to outpatient clinic when referral documented palpable mass.,Those in the low-risk arm who had a qFIT value of 80 µg/g or above had a CT mini-prep added. Given the paucity of data in symptomatic populations at the start of the pandemic, the decision to use the threshold of 80 μg/g was pragmatic and based on the Scottish bowel-screening guidelinesThe pathway was dynamic to the return of some diagnostics after June 2020 and changes were aimed at improving CRC detection and safeguarding; patients with qFIT values greater than 80 µg/g or equivocal CT findings underwent colonoscopy and patients with a qFIT value between 10 and 79 µg/g underwent CT colonography (CTVC); CT mini-prep was used as a safety-net measure for those who returned two qFIT values less than 10 μg/g.The outcomes that were investigated in this evaluation were the risk of a missed CRC diagnosis and the proportion of referrals requiring a colonoscopy or CTVC. Colonoscopy and CTVC were considered equivalent for the purpose of this study. Risk of a missed CRC was considered to be a sufficient proxy measure of patient outcomes while number of colonoscopies or CTVCs are the key resource constraint in the service.Decision-tree models were developed based on plausible and potentially desirable alternative diagnostic pathways. In all cases the diagnostic pathway was modified to include triage testing . The pathway scenarios included were: option 1 – single qFIT; option 2 – two qFITs; option 3 – single qFIT and a CT mini-prep; option 4 – two qFITs and a CT mini-prep (enriched pathway). These scenarios were compared with the standard diagnostic pathway (colonoscopy or CTVC with assumed 95 per cent sensitivity at best)Payoffs at the terminal nodes of the decision trees were limited to two binary indicators: whether or not a missed cancer occurred and whether or not a colonoscopy or CTVC was required. Node probabilities were defined based on local audit data in NHS Lothian as outlined above.versus worst-case) scenarios were made by expert opinion based on available data by the research team . As an example, CT mini-prep was used to rule out gross pathology and was not directly compared against the current standard diagnostic tests (colonoscopy or CTVC) to diagnose CRC, thus negative CT mini-prep had to be assumed truly negative in the best scenario  although the authors also considered the worst-case scenario of sensitivity of 50 per cent.Sensitivity and specificity of colonoscopy, qFIT and CT mini-prep were obtained from the audit, SCAN data, literature and, if missing, assumptions on reasonable , some estimates and assumptions were required to construct models.All base case parameter values are detailed in GP referrals are made in three categories: urgent suspicious of cancer (USOC), urgent and routine. To estimate the target CRC-detection rate of the COVID-adapted pathway, the prevalence of CRC from GP referrals was calculated from the number of cancers diagnosed against the number of all GP referrals received during same months in the previous years . The prevalence was calculated per all referrals and per USOC and urgent combined, as the latter is most likely to match the incoming GP referrals and produce the highest yield of CRC.,Incremental diagnostic yield was defined as the probability of a second qFIT value of 10 µg/g or greater when the first qFIT value was less than 10 µg/g. Incremental true positives and incremental false positives were derived from the incremental diagnostic yield under the assumption that cases and non-cases were equally likely to convert. Sensitivity and specificity of CT mini-prep were estimated from local radiologists' estimates (sensitivity 75–80 per cent) and available data in literature Decision-tree model outputs were the percentage risk of missed cancer for cancer cases, number of missed cancers per 1000 in the pathway and number of colonoscopies required per 1000 in the pathway.http://www.R-project.org) with package ‘data.tree’.The analysis was performed using software R version 4.0.0 , the average rate of CRCs detected for all the referrals  was 3.8 per cent. When the referrals were restricted to a combination of USOC and urgent referrals (excluding bowel cancer screening), the average rate was 9.4 per cent.,Based on these figures, 3.8 per cent has been set as the lowest detection rate, 10.0 per cent as highest possible rate (and worst-case scenario) and 6.6 per cent (average of 3.8 and 9.4 per cent) as likely local CRC detection rate from combined USOC and urgent referrals, taking into account data from the literature (around 7 per cent)Some 325 CT mini-preps were performed in the COVID-adapted pathway. Equivocal CTs were examined by two consultant radiologists. The sensitivity of detecting cancer was between 71.4  and 85.7 per cent , assuming all normal CTs were truly normal. With these caveats in mind, the sensitivity of CT mini-prep included in the model sensitivity analysis was set between 50 (worst-case scenario) and 85.7 per cent (base case scenario).,The sensitivity of the qFIT for detecting cancer at the threshold of 10 µg/g was set as 82.5 per cent for the decision-tree model based on the authors’ local audit data of symptomatic patients over the previous year, which fluctuated between 80 and 85 per cent, in keeping with published data of qFIT sensitivity for symptomatic peopleTable 2 and Fig. 1.Risk of missed cancer and rate of colonoscopies for each of the alternative pathway scenarios are reported in The risk of missing a cancer, if present, was as high as 20.2 per cent with a single qFIT at the threshold of 10 µg/g used as the sole triage test. This is equivalent to approximately 13 missed cancers per 1000 patients that are evaluated using this diagnostic pathway. Using both a second qFIT and a CT mini-prep as add-on tests, the estimated risk of missed cancer reduced to 6.49 per cent, or approximately four missed cancers for every 1000 patients on the pathway. The use of CT mini-prep as an add-on test is predicted to decrease the number of missed cancers substantially compared with adding a second qFIT. The trade-off for the use of the add-on tests is an increased rate of colonoscopy or CTVC, from 287 for a single qFIT to 418 for double qFIT and CT mini-prep combination, per 1000 patients on the pathway.Figure2 shows the risk of missing cancer across a range of CT mini-prep test sensitivity values. As expected, the prevalence of CRC among the referred patients influences the rate of missed cancers and number of colonoscopies or CTVC required. Diagnostic outcomes under scenarios of 3.8 and 10 per cent prevalence are displayed in Table 3. Although missed CRCs are increased substantially at higher prevalence, the number of colonoscopies or CTVCs shows only a modest increase as the large majority of patients in the pathway do not have CRC.This scenario explored the impact of assuming CT mini-prep sensitivity to be as low as 50 per cent. This would result in an increase in the probability of missing a cancer to 12.61 and 10.24 per cent for options 2 and 4 respectively (8.32 and 6.75 missed cancers per 1000 patients on the pathway). This is substantially worse than the base case value although still fewer missed CRCs than under the double qFIT scenario (option 3). There would also be a slight decrease in the rate of colonoscopies or CTVCs to 335 and 400 per 1000 for options 2 and 4 respectively.This COVID-adapted pathway is an example of adding a triage test sequence to an existing definitive diagnostic testVarious measures have been proposed to mitigate risks of missing CRCs and rationing available resources during the COVID pandemic. Understandably, there have been limited real-time data to guide whether adoption of such strategies does indeed mitigate against missing CRCs and, importantly, what residual risks are present for patients referred on such alternative pathways.Although qFIT has been advocated by many to be a useful tool to ration limited diagnostics, caution was used in adopting the single qFIT approach due to its low sensitivity as a rule-out test in symptomatic patients. The present study shows the incremental value of adding standard CT with minimal preparation as it drives down the risk of missing CRC by 65 per cent, compared with a single qFIT.versus 5 per cent for colonoscopy). Prior to the pandemic the commonest triage outcome was to offer ‘direct to test’ with minimal gatekeeping. The data presented here suggest one could further reduce the number of colonoscopies required without compromising CRC detection rate. Robust analysis of cost-effectiveness will require additional data regarding impact on health resource use in the long term, such as repeated presentation, follow-up investigations and treatment costs.The addition of a second qFIT does increase access to subsequent investigations, but there have been no CRCs detected in the 6.9 per cent of patients who had a positive result following an initial negative result and hence true enrichment for cancer detection cannot be calculated at this stage. Therefore, determining the value of performing double qFIT needs a longer follow-up and further data collection. Risk calculation could be further optimized by incorporating nodal information such as existing risk factors for CRC , blood results  and itemized symptoms. The combination of double qFIT and CT mini-prep is substantially cheaper compared with a single colonoscopy with a similar risk of missing cancer (6.5 per cent The data give transparency in terms of risks with each scenario used and the reasoning required to reach a decision about the most appropriate form of triage, given the constraints faced by the service. This information is also useful to inform GPs and patients appropriately about this alternative pathway as a reasonable option in the current resource-limited situation and give reassurance.The major limitation of this analysis is lack of a head-to-head comparison of alternative and standard diagnostic pathways. In the absence of a randomized controlled trial or comparative study, data were retrieved from a large local audit investigating the use of qFIT for symptomatic patients with established outcomes by standard diagnostic tools. Clinical decisions must be made amidst a resource-constrained situation during the ongoing pandemic, thus the residual risks were calculated based on modelling assumptions to monitor the performance of the adapted pathway. Direct evidence could be obtained in the future from data collected in a prospective cohort study design in which patients receive all triage tests as well as the reference diagnostic test. An important caveat to the present approach, which mitigated risk by achieving similar CRC detection rates compared with previous years, was that referrals were decreased by 50 per cent, highlighting the likelihood of undetected cancersTriage using qFIT alone leads to an unacceptably high rate of missed CRCs. This may be reduced effectively by using CT mimi-prep as an add-on test for triage to colonoscopy or CTVC. There is a need to gather more data on the diagnostic accuracy of the combined qFIT and CT pathway to validate this approach and provide confidence in the protocol.zrab056_Supplementary_DataClick here for additional data file.


Triclosan (TCS) is a phenolic compound with broad-spectrum antimicrobial action that has been incorporated into a variety of personal care products and other industry segments such as toys, textiles, and plastics. Due to its widespread use, TCS and its derivatives have been detected in several environmental compartments, with potential bioaccumulation and persistence. Indeed, some studies have demonstrated that TCS may act as a potential endocrine disruptor for the reproductive system. In the current study, we are reporting on the results obtained for male rats after a two-generation reproduction toxicity study conducted with TCS. Female and male Wistar rats were treated daily by gavage with TCS at doses of 0.8, 2.4, and 8.0 mg/kg/day or corn oil (control group) over 10 weeks (F0) and over 14 weeks (F1) before mating and then throughout mating, until weaning F2 generations, respectively. TCS exposure decreased sperm viability and motility of F1 rats at the dose of 2.4 mg/kg. The effects of TCS on sperm quality may be related to the exposure window, which includes the programming of reproductive cells that occurs during fetal/neonatal development. Endocrine disruptors are referred to as environmental contaminants, described as an exogenous substance or mixture that alters function(s) of the endocrine system with adverse health effects in an intact organism, its progeny, or (sub)populations , 2. SeveTriclosan (TCS) is a trichlorinated phenoxyphenol with broad-spectrum antimicrobial action that has been incorporated into a variety of consumer products including soaps, hand sanitizers, toothpaste, mouthwash, and lotions, as well as being used in other consumer products such as toys, the textile industry, and plastics –8. The sThe worldwide annual consumption of TCS in 2015 was approximately 4,600 tons . BecauseThe continuous detection of TCS including its degradation products in the environment –22 raiseIn vitro, TCS exposure suppressed testosterone production in two Leydig cell-based assays  the widespread use of TCS and the risks involving its environmental exposure over the decades; (2) the potential TCS toxicity to the reproductive system and organism development; and (3) that the exposure to endocrine disruptors during early development may cause a growing set of diseases , 33, whiThe two-generation reproduction toxicity study was based on the OECD Guideline for Testing of Chemicals 416 , and theTCS was obtained from Vivimed Labs Limited   and dissolved in corn oil (vehicle), before being administered orally, by gavage, in a volume of 2.5 ml/kg.The description of the dose obtention was published previously in Pernoncini et al. . AccordiSeventy-five-day-old male and female Wistar rats from the colony of the State University of Londrina were maintained in the animal facilities of the Department of Physiological Sciences in a controlled environment at a temperature of 21 ± 2°C, 12 h light/dark cycle (lights on at 6:00 a.m.), with free access to regular lab chow  and water. Animals were housed in collective polypropylene cages (29 × 18 × 13 cm) with wood shavings as bedding and were mated after 1 week of acclimatization. Two females were mated with one male to obtain the F0 generation. The day of birth was considered postnatal day (PND) 0. All animal procedures were approved by the State University of Londrina Ethics Committee for Animal Research (CEUA/UEL: 283.2015.27).Male and female Wistar rats were used as the F0 generation (15–17 rats/sex/group). On PND 49, animals (both sexes) were randomly allocated, stratified by weight, to one out of three treatment groups to receive oral TCS doses of 0.8, 2.4, and 8.0 mg/kg, in addition to the control (CTR) group, treated with vehicle alone. Animals were treated once a day (11:00 a.m.–1:00 p.m.) for 70 days in order to cover at least one complete spermatogenic cycle and several complete cycles of oogenesis. At PND 120, animals were mated (as described below). Treatment continued throughout mating as well as pregnancy and lactation for the F0 females , while in F0 males it continued for 20 days after mating in order to replace the spermatic stock.For mating, F0 animals were cohabitated overnight daily  until a positive pregnancy diagnosis. Females with sperm observed in the vaginal smear were considered as day 0 of the presumed gestational day (GD) and housed individually. Pregnant females were allowed to deliver their pups naturally. The day on which parturition was completed until 1:00 p.m. was designated as lactation day (LD) 0 for dams and PND 0 for pups. All live pups (F1 generation) were identified by skin tattoo on PND 1. Litters were culled on PND 4 to 8 pups, keeping four males and four females whenever possible and identified by ear marking at weaning (PND 21). Three male pups were used for the F1 analysis: one to evaluate the sexual development, physical development, and neuromotor development; other for motor activity; and another one for mating and sexual parameters.The third male and one female (not sibling) were selected to parent the F2 generation. They were gavaged, following the same treatment protocol already described, from PND 22 (24 h after weaning) up to weaning of the F2 offspring, in this way, covering the mating period, which occurred around PND120, gestation and lactation. The F2 pups were exposed to TCS through F0 and F1 generation, placenta, and lactation and were evaluated only until weaning. The animal health status and clinical signs  were checked daily.The females from F0, F1 and F2 were used in another study to determine the reproductive toxicity to TCS exposure, and the results have already been published in Montagnini et al. . The datBody weight was measured every 3 days, from the beginning of the dosing period  to the day before mating (PND 120), considered as the pre-mating period . Food consumption was obtained weekly during this period.All behavioral assessments were performed in adult rats (beginning on PND 120)  during the dark phase of a reversed light/dark cycle, under dim red light. The animals were allowed 15 days of adaptation to the reversed light/dark cycle before the beginning of evaluation. The observations always started 2/3 h after the onset of darkness and were recorded by a video camera, linked to a monitor in an adjacent room.For the evaluation of copulatory behavior, each male was placed into a Plexiglass cage with dimensions 20 × 40 × 50 cm (height × width × length), and after 5 min, a non-treated female in natural estrous was introduced into the cage. For 30 min, the latencies and number of intromissions and ejaculations were observed as described previously in Gerardin et al. . If a ma2. The small arenas are diagonally opposed to each other, and the communication with the main arena is closed with a wire mesh. For the test, an estrous female  was placed in one of the small arenas and a sexually active male  was placed in the other arena. The floor of the main arena had two 25 cm2 divisions (zones) in front of each small arena opening, named female and male incentive zones, respectively. The experimental male was placed in the center of the main arena and observed for 20 min. The number of visits and the total time spent visiting each zone were quantified, and a preference score was calculated as  × 100  was used, with two openings that lead to two small arenas of 25 cms) × 100 .The F1 males used for sexual behavior evaluation were euthanized by decapitation 20 days after mating . Blood was collected for testosterone quantification. The testis (pair), vas deferens, epididymis (left), ventral prostate, liver, kidneys (pair), adrenal glands, and seminal vesicle (pair) (without the coagulating gland and full of secretion) were removed and their weights determined. The right testis and epididymis were frozen at −80°C for sperm counting, and the left testis was collected for histomorphometric analysis. The right vas deferens was used for the analysis of motility, viability, and sperm count and the left for sperm morphology.Blood samples were collected from the abdominal aorta into syringes containing heparin, then centrifuged , and the plasma frozen until assayed. Plasma testosterone quantification was measured by chemiluminescence microparticle immunoassay  according to the manufacturer’s instructions, with an intra-assay coefficient of variation and minimum sensitivity of the assay of 4.6% and 0.15 nmol/L, respectively.Sperm motility analysis was performed according to Perobelli et al.  and FavaFor sperm viability, the one-step eosin–nigrosin staining technique was used . A total6/ml) was performed in a Makler chamber . Sperm was counted in 10 different squares chosen at random, in four different fields, and the average was obtained  and sperm in the caput/corpus epididymis and cauda epididymis were assessed as previously described by Robb et al.  with ada2; R = tubular diameter/2, according to the formula: LST = TSV/πR2)  was promptly dissected, weighed, and fixed by immersion in Bouin’s solution for 24 h before being stored in ethanol at 70°. The testis was cut into tissue fragments and routinely processed for embedding in Paraplast™ blocks. Six semi-serials sections of 7 µm separated by a distance of 50 μm per section were obtained from each animal and stained with hematoxylin-eosin. The diameter of the seminiferous tubules was measured at 100× magnification using an ocular micrometer. Fifteen cross-sections of tubule profiles that were either round or nearly round were chosen randomly and measured for each animal. The volume densities of testicular tissue components were determined by light microscopy using a 100-intersection grid placed in the ocular of the light microscope. A total of 10  randomly chosen fields were scored for each animal at 400× magnification. The volume of each component of the testis was determined as the product of the volume density and testis volume. For subsequent stereological calculations, the specific gravity of testis tissue was 1.0 . To obtaTSV/πR2) , 54.All offspring of F1  and F2  generations were examined as soon as possible on the day of birth (PND 0) to determine the number of liveborn and stillborn per litter; the pups were sexed based on the anus to genital tubercle distance. Pups were weighed on PNDs 1, 4, 7, 14, and 21. Pup health status and clinical signs were checked at least once daily throughout lactation.Sexual development data were obtained from one randomly selected male of each litter from F1  and F2  generations. The anogenital distance  was obtained through a Vernier caliper on PNDs 1, 4, and 21. Relative AGD was normalized through its division by the cube root of body weight . MoreoveThe age at complete preputial separation was recorded to investigate puberty onset and was recorded only from F1 males selected for mating.The following physical developmental parameters were observed: pinna detachment  (from PND 2), incisor teeth eruption (from PND 6), and eye opening (from PND 10) . Age at eye opening and ear detachment was recorded when both eyes and ears were completely opened. The incisor eruption was examined daily to determine the first day of the bilateral appearance of the upper incisors.The same pup selected for sexual and physical development evaluations was used for all neuromotor behavior assessments . The dependent variable analyzed for each test was the day of first achieving either maturity of the reflex or the conditions listed below.The neurobehavioral assessments were evaluated following the OECD/OCDE Guideline 426 for Developmental Neurotoxicity Study  by a traRighting reflex: Starting on PND 5, the pup was placed on its back on a flat surface and allowed to right itself. Time to regain normal position was recorded. This reflex was completed if the pup performed this response within 10 s.Negative geotaxis: Starting on PND 5, each rat was placed on an inclined wire mesh ramp (angle of inclination from the base: 45°) with the head facing down. The reflex was acquired when pups performed a 180° body rotation and when they could climb up within 30 s.The righting reflex is a test used to investigate the reflection of sensorimotor coordination, while the negative geotaxis reflex evaluates the cerebellar integration. In rodents, these reflexes arise on average at PND 5 and at PND 7, respectively .Motor activity: Motor activity behavior of offspring on PNDs 13, 17, and 21 was evaluated in an open-field arena in F1  and F2 pups . The apparatus consists of a circular wooden surface with a diameter of 39 cm, surrounded by a wooden wall 28 cm in height. The surface is painted white and divided into 19 similar parts by concentric circles and straight segments. Each animal was placed in the center of the arena and its locomotion evaluated  during a 10-min session . After tpost-hoc test, with data being presented as mean ± standard error of the mean (SEM). Conversely, for other variables the Kruskal-Wallis complemented with Dunn’s test was performed with the presentation of data as median (1st and 3rd quartiles). Analysis of covariance (ANCOVA) was used to detect the effect of the treatment on organ weight using the final body weight as the covariate. However, since the ANCOVA and ANOVA provided similar results, the ANOVA results are reported. For pup body weight, relative AGD, and motor activity, a repeated measures ANOVA (RMANOVA) was applied with day as the within-subject factor and treatment as the between-subject factors. The Chi-Square test was used to examine differences in categorical variables . Differences were considered significant if p < 0.05. All statistical analyses were performed using SPSS .An exploratory analysis was conducted to evaluate the normal distribution (Shapiro-Wilk test) and homogeneity of variance (Levene’s test) of each variable. In the absence of normal distribution and/or homogeneity of variance, variables were transformed in order to achieve the criteria for parametric analysis. Variables that presented normal distribution and homogeneity of variance were analyzed by ANOVA complemented with the Bonferroni No mortality, morbidity, or general signs of toxicity such as changes in behavior , neurological changes , or autonomic signs  were observed during the treatment period. = 0.239, p = 0.869]. No treatment effect of TCS was observed in the mean feed consumption of F0 and F1 generation groups during these periods (data not shown).Body weight gain was calculated from the beginning of the dosing period (PND 22) to the day before mating (PND 120). As described by ANOVA, the chronic administration of TCS did not affect body weight gain (g) during the pre-mating period of F1 rats  , AGD, , organ weights , or plasma testosterone quantification (ng/dl) .The final body weight (g) at PND 140, AGD, organ weights, and plasma testosterone quantification of adult males of the F1 generation are presented in (3.33) = 5.935, p = 0.003] and the percentage of mobile sperm  in males of the TCS 2.4 group, compared to the control animals  = 0.979, p = 0.416], interstitial content volume , seminiferous tubules volume , diameter of seminiferous tubules , and total length of seminiferous tubules  were detected by ANOVA complemented with Bonferroni. The testis parenchyma structure of rats on PND 140 of the F1 generation is shown in The testicular histomorphometry results of the F1 generation are given in Pup body weights (litter mean) of F1 and F2 generations during the first 3 weeks of life are illustrated in The number of pups and sex ratio (M:F) were not affected by TCS treatment (data not shown). Relative AGD (mm/g1/3) of F1 and F2 male pups at PNDs 1, 4, and 21 were not influenced by TCS (p > 0.05) as indicated by RMANOVA. Nipple retention  on PND 12 was 100% in all experimental groups of both F1 and F2 generations . ANOVA was performed to compare the age (day) in which preputial separation occurred in F1 males, and no difference was observed among the groups (p > 0.05) (data not shown).Data from physical and neuromotor parameters of F1 and F2 pups are presented in Data from the behavioral evaluation of males from F1 and F2 generations in the open-field  on PNDs 13, 17, and 21 are given in Rodent and human exposure to TCS is frequently associated with decreased thyroxine levels with consequent impairments to reproductive and developmental health , 59. TheIn this study, male rats exposed to TCS throughout the intrauterine development and lactation until adulthood did not present alterations in body weight gain. This result is consistent with those observed in Wistar rats treated with TCS at doses ranging from 3 to 300 mg/kg , 28. Morin vitro data indicate that TCS may exert adverse effects on reproductive functions. In rat testicular tissue the TCS exposure reduced the activity of steroidogenic enzymes: 3β- hydroxysteroid dehydrogenase and 17β- hydroxysteroid dehydrogenase , it was observed that, in the epididymis, the TCS has a longer retention time and a longer elimination half-life compared to testis and prostate plasma, showing a tendency for the accumulation of this compound in the epididymis and consequent histopathological damage  of the testicular parenchyma of adult rats of the F1 generation. In Priyanka et al. , maternal damage . Furtherl damage , reinforRegarding sexual development, exposure to TCS during intrauterine development, lactation, and, subsequently, throughout development did not alter the AGD of F1 and F2 males, which is a marker of fetal androgen action , and alsIn this study, TCS exposure did not induce significant alterations in physical, neuromotor, and motor activity development, in both F1 and F2 generations. Neuromotor evaluation (open field) during the lactation period allows identification of possible neurobehavioral alterations in the early life of these animals. These evaluations are important because the development of the offspring’s nervous system might be indirectly affected, since environmental toxicants are capable of inducing non-specific effects that include alterations in hormone release and/or the nutritional status of dams . These dIn summary, the treatment and maternal (placenta and lactation) exposure to TCS compromised sperm parameters in rats of the F1 generation, observed by a decrease in motility and viability of sperm at 2.4 mg/kg. This is the first study to examine the long-time effects of exposure to TCS through germ cells, placenta, lactation, and oral treatment on reproductive potential, including sexual behavior, and physical and neuromotor development in male Wistar rats. These results emphasize the importance of studying the safety of exposure to TCS on reproductive function for the population. Although the TCS toxic effects on sperm quality only occurred at the intermediate dose (2.4 mg/kg), it is important to clarify that the doses used in this study were based on the maximum limits of exposure to TCS in the diet recommended by regulatory agencies. As previously stated, the TCS 2.4 group received a higher dose (compared to TCS 0.8 group) to compensate for potential intraspecies variations, and these animals were susceptible to TCS sperm toxicity. A further epigenetic investigation is needed to clarify how exposure to TCS during important development windows affects the sperm quality of rats.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the State University of Londrina Ethics Committee for Animal Research (CEUA/UEL: 283.2015.27).BM conceived and designed the study. SF helped with data obtention and played an active role during manuscript drafting. KP and MM performed experiments of F1 and F2 generations. MP performed histological analysis. NC performed sperm parameters evaluation. EM contributed with the technical and data review. JA-F quantified plasmatic testosterone, and DG supervised and coordinated the study. All authors listed have contributed to the work and approved the final version.This work was supported by CAPES  and CNPq (454499/2014-0).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Multifunctional magnetic nanowires (MNWs) have been studied intensively over the last decades, in diverse applications. Numerous MNW-based systems have been introduced, initially for fundamental studies and later for sensing applications such as biolabeling and nanobarcoding. Remote sensing of MNWs for authentication and/or anti-counterfeiting is not only limited to engineering their properties, but also requires reliable sensing and decoding platforms. We review the latest progress in designing MNWs that have been, and are being, introduced as nanobarcodes, along with the pros and cons of the proposed sensing and decoding methods. Based on our review, we determine fundamental challenges and suggest future directions for research that will unleash the full potential of MNWs for nanobarcoding applications. Initially, barcodes were invented for the authentication of products in anti-counterfeiting, which is of the foremost importance due to the continuous growth of non-transparent trading ,2,3,4. NIn the big picture, there are the following three essential merits for the ideal nanobarcode: (1) expandable encoding, (2) secure sensing, and (3) reliable decoding, as shown in Recently, MNWs became the center of the research in nanobarcoding because of the revealed potential for making the next generation of nanobarcodes and/or biolabels ,24,25,26Each magnetic nanowire (MNW)-based nanobarcode is made of a collection of MNWs, where the magnetic properties of each MNW and the intra-magnetic interactions can be used for encoding ,41. The The hysteresis loop measurement is the most popular and fastest method for magnetic property extraction, and it provides the saturation magnetization and the coercivity of any sample. Tailoring the saturation magnetization is best to be conducted using magnetic components with high saturation magnetization, such as iron, if the other components are non-magnetic ,58,59. ICoercivity is another important magnetic property that determines how resilient the spins are against changing their direction. Simply, coercivity is the required magnetic field to rotate the magnetization 90 degrees, or for very anisotropic samples, it is the field required to produce equal “up” and “down” spins. The simplest way to tailor the coercivity is probably to vary the MNW sizes ,76,77, aInducing any chemical or physical changes/mismatches that facilitate or hinder the switching direction of the spins would result in tailoring the coercivity ,82. An eThe first-order reversal curve (FORC) method is another DC measurement that has been broadly used for the qualitative, and partially quantitative, description of magnetic signatures ,98,99, aWhen plotting each pixel , in terms of the reversal and applied field, the reversal field is the field in which magnetization switches from +1 to −1, while the applied field is the field in which the magnetization switches reversely. In other words, half of their difference  is the coercivity, and half of their summation  is the interaction field ,100,102.As mentioned earlier, the FORC heat-maps have been broadly used as qualitative descriptions of MNWs magnetic signatures. For quantitative description, the heat-maps are projected onto the coercivity and interaction fields in order to calculate the coercivity and interaction field distributions, and to be used as quantitative signatures ,107. ThiAmong all of the approaches to speed up the FORC measurements, the projection method was proposed, to significantly accelerate the sensing of magnetic nanobarcodes signatures, particularly for biolabeling and nanobarcoding ,27,34. AIt should be emphasized that the projection method measures the irreversible switching field distribution at the reversal field. The advantages of the irreversible switching field distribution for nanobarcoding have attracted attention towards measuring the irreversible switching field distribution at zero field, which is also known as the backward remanence measurement ,6, as shAC measurements apply an alternating magnetic field and measure the response of magnetic nanobarcodes at different frequencies or a biased magnetic field. The two widely used AC measurements are magnetic particles spectroscopy ,119,120,Another AC measurement for sensing magnetic nanobarcodes is magnetic resonance spectroscopy ,128, alsAs discussed in the previous section, there is a trade-off between fast sensing and reliable sensing. A solution for avoiding this trade-off is to reliably sense multiple magnetic nanobarcodes, to speed up the decoding by reducing the number of required readouts/measurements. In contrast to the huge progress in the encoding and sensing of magnetic nanobarcodes, the reliable decoding of them has not received much attention, even though it is crucial for the commercial transition ,6. FurthFor reliable decoding of multiple unknown MNW-based nanobarcodes, it is necessary for the magnetic nanobarcodes to have distinct features, with minimum overlapping. This requirement discards the hysteresis loop measurement as it only provides single values, for example, saturation magnetization or coercivity. Aside from the fact that the saturation magnetization and coercivity are well known and have strong magnetic signatures for encoding, they are insufficient for reliable decoding, especially when there is more than one nanobarcode at the readout ,6,7. ForThe key for the reliable decoding of multiple nanobarcodes is that the readout signal of a combination must be a linear superposition of the individual components. This is usually achievable because the interwire distance between the MNWs  in a nanobarcode is several orders of magnitude smaller than the distance between the nanobarcodes . This allows the readout signal to be a linear superposition of the signatures of the composed nanobarcodes/subdivisions. Indeed, the challenge is to determine the number of nanobarcodes; 1, where superscript one indicates N = 1. Next, N is increased to 2 and the new optimum RMS error, RMS2, is calculated. Then, RMS2 is compared with RMS1 to determine how much the RMS error decreased, by increasing N from 1 to 2. If the reduction meets the cutoff, then there are at least two nanobarcodes at the readout (N ≥ 2), as shown in 3 and RMS2 must be considered, and their ratio must be compared with the cutoff value, as in 3 compared to RMS2 was not sufficient, the decoding process can be terminated, because it would appear that only two nanobarcodes were present at the readout (N = 2). This process must be continued until the ratio of RMSN-to-RMSN-1 is no longer smaller than the cutoff value. The main drawback of this technique for reliable decoding is finding the correct value for the cutoff. For example, as the number of nanobarcodes at the readout increases, the magnetic signatures start overlapping, which makes the decoding difficult. It should be emphasized that this drawback is not limited only to magnetic nanobarcodes, as the reliable decoding of any nanobarcodes suffers from this problem. To resolve this problem, we proposed two alternatives. The first alternative is to use a floating cutoff value, which is a function of the predicted number of nanobarcodes. The second alternative, which could be a more effective approach, is to eliminate the need for a cutoff, which could be accomplished by using the artificial intelligent (AI) or the machine learning (ML) approaches. To accelerate the transition of MNW-based nanobarcodes to real-life applications, the reliable decoding of multiple nanobarcodes demands a huge amount of attention, with many research opportunities in computer science and signal processing domains, which are expected to flourish soon.Though considerable progress and advances have been recently made toward the encoding/sensing/decoding of MNW-based nanobarcodes, there are still challenges for their use in nanobarcoding applications that need to be addressed to achieve practical translation. The extremely important merits of promising nanobarcodes are as follows: (1) expandable encoding capability, (2) fast sensing with minimal background noises, and (3) reliable decoding of multiple nanobarcodes simultaneously present at the scanner. The encoding capability and sensing merits are inherently correlated, as the former is the targeted magnetic property/signature and the latter is the magnetic measurement method for sensing/measuring the targeted property. The main considerations that must be taken into account when choosing the proper property/signature and sensing/measurement are the stability over time, expandability, and the speed and reproducibility of the signature, in addition to the cost, ease of use, and portability of the sensing instruments. These criteria make many magnetic signatures unusable, and leave a few options available, which can be categorized based on the type of sensing methods, which are DC and AC methods. Basically, the DC sensing methods are more reliable and simpler, but slower than the AC sensing methods. More importantly, the DC methods intuitively provide sensing of the hidden MNW-based nanobarcodes, with higher signal-to-noise ratios compared to the AC methods.The need for faster sensing, based on DC methods, that provides distributions, instead of single values, to achieve reliable decoding, eliminates the hysteresis loop and first-order reversal curve (FORC) methods, which are brief and slow, respectively. Among all, the projection method and backward remanence deem more promising, as they are as fast as the hysteresis loop method, companied by detailed analyses similar to the FORC method. To further enhance the rapid authentication, it is essential to be able to reliably decode the readout signals from multiple nanobarcodes simultaneously present at the scanner. This requirement demands other fields, such as signal processing from electrical engineering and machine learning or artificial intelligence from computer science, into magnetism, to facilitate the realization of MNW-nanobarcodes translation to daily applications. Indeed, despite the huge progress in the encoding and sensing of MNW-based nanobarcodes, the reliable decoding of multiple MNW-based nanobarcodes is still in its rudimentary stage and requires much exploration.