Escherichia coli infection is a main cause of diarrheal disease in endemic countries. The characterization of the immune responses evoked by natural infection can guide vaccine development efforts. (2) Methods: A total of 617 adult and 480 pediatric diarrheal patients were screened, and 43 adults and 46 children (below 5 years of age) with an acute ETEC infection completed the study. The plasma samples were analyzed for antibody responses against the ETEC toxins. (3) Results: Heat-stable toxin (ST)-positive ETEC is the main cause of ETEC infection in adults, unlike in children in an endemic setting. We detected very low levels of anti-ST antibodies, and no ST-neutralizing activity. However, infection with ETEC strains expressing the heat-labile toxin (LT) induced systemic antibody responses in less than 25% of subjects. The antibody levels against LTA and LTB, as well as cholera toxin (CT), correlated well. The anti-LT antibodies were shown to have LT- and CT- neutralizing activity. The antibody reactivity against linear LT epitopes did not correlate with toxin-neutralizing activity. (4) Conclusions: Unlike LT, ST is a poor antigen and even adults have low anti-ST antibody levels that do not allow for the detection of toxin-neutralizing activity.A sero-epidemiology study was conducted in Dhaka, Bangladesh between January 2020 and February 2021 to assess the immune responses to ETEC infection in adults and children. (1) Background: Enterotoxigenic  Escherichia coli (ETEC) represents a major cause of diarrhea among children <5 years living in endemic countries and also travelers to endemic regions.Despite the improvements over the last few decades in drinking water quality, sanitation, and the implementation of current prevention and treatment interventions, diarrheal diseases remain a major cause of illness, death, and developmental disabilities such as stunting and cognitive development especially among children in low- and middle- income countries (LMICs) under five years of age ,2,3,4. SSalmonella Typhi (against typhoid). In addition to the use of proven measures, a comprehensive approach to address diarrhea is urgently required, including the development of innovative tools to fill current gaps in effective prevention strategies. Vaccines in particular are a major tool for the prevention of diarrheal diseases ,6 but cuETEC pathogenesis is dependent on the production of diarrheagenic toxins: the heat-labile toxin (LT) and/or the heat-stable toxin (ST). LT and ST cause alterations in the ion and fluid transport of epithelial cells, which results in increased water secretion into the gut (diarrhea). This can contribute to severe dehydration with detrimental consequences especially in young children living in countries without an appropriate healthcare infrastructure. LT is a multi-component bacterial toxin assembled from two separately expressed subunits . FollowiST is an 18 or 19 amino acid-long cyclic peptide  that binds to its receptor, guanylate cyclase C (GC-C). This activation coincides with elevated cGMP production and leads to increased water retention in the gut (diarrhea). ST is a challenging vaccine antigen due to its small size and the consequent low immunogenicity, as well as its complex tertiary structure [ETEC-induced diarrhea is mainly attributed to the effects of the LT and ST toxins. The antibodies neutralizing these ETEC toxins can be considered protective, although it is not known what levels of such antibodies are sufficient. Adults living in endemic regions acquire protective immunity due to repeated exposure, while young children are the most susceptible. We herein characterized the anti-toxin antibody profiles in plasma samples of infected adults and children living in Bangladesh, which is endemic for ETEC infections. Our goal was to assess the toxin-neutralizing potency of plasma from the acute infection phase and convalescence. This information is expected to guide vaccine efforts and approaches against ETEC infection.This study was approved by the Ethical Review Committee (ERC) of the icddr,b and conducted using Good Clinical Practice (GCP) and in accordance with the World Medical Association (WMA) Declaration of Helsinki. For screening diarrheal patients, the medical team identified and selected potential patients with typical symptoms of diarrhea from the selected health facilities in Mirpur as well as icddr,b Dhaka hospital. In general, patients with watery stool were examined as suspected cases of ETEC diarrhea. Stool samples from these patients were collected to detect ETEC by culture followed by PCR. A signed and dated informed consent document was obtained from adult participants or parents of child participants before initiating any study-specific procedure.Escherichia coli and absence of significant comorbid conditions. 2. Male or female adult (18–45 years) and children (up to 5 years) patients. 3. Free of concurrent infections. 4. Adult participants or father/mother of child participants properly informed about the study, able to understand it and sign the informed consent form. 5. Available for the entire period of the study and reachable by study staff throughout the entire follow-up period.Inclusion criteria: 1. A positive stool culture for enterotoxigenic Exclusion criteria: 1. History of immunization with experimental ETEC or Shigella vaccines. 2. Pregnant women. 3. Presence of a known significant medical or psychiatric condition that in the opinion of the investigator precludes participation in the study. 4. Those receiving immunosuppressive therapy. 5. Prior receipt of a blood transfusion or blood products, including immunoglobulins. 6. Evidence of current illicit drug use or drug dependence (adult participants only). 7. Any condition which in the opinion of the investigators puts the subject at risk of noncompliance with the protocol. Blood samples from patients were collected at enrolment (day 2/3 after onset of diarrhea), and follow-up visits at day 7 ± 1 and day 30 ± 4 and once from healthy adult participants. Approximately 10 mL of blood was collected from adults and 5 mL from children. Blood samples were collected in a heparinized tube, and immediately transported to the icddr,b laboratory, maintaining cold chain. Plasma samples were separated from blood and stored in aliquots at −80 °C.2SO4, and OD450nm was measured with an EON plate reader . Alternatively, ABTS substrate  was used and OD405nm was measured with a Synergy HTX (BioTek) plate reader. Human serum samples from non-endemic healthy adults without any history of an ETEC infection were used as a negative control. The determined end point titer corresponds to the highest dilution factor giving a signal value above the mean signal of the negative control multiplied by the mean signal of the sample at the corresponding dilution. Alternatively, titer was determined using Gen5  software.ELISA plates with 96 wells or Streptawell ELISA plates were coated with the respective antigen: heat-stable toxin , heat-labile toxin B subunit , heat-labile toxin A subunit , or Cholera toxin B subunit . Serial dilutions of plasma samples were run together with negative controls on each plate. Goat F(ab’)2 anti-human IgG-HRP  or Goat Anti-Human IgA-HRP (SouthernBiotech) was used and detected either with Sureblue TMB , blocked with HN12S protein [2) for 3 h or 1 h for LT- or ST-assays correspondingly. Supernatants were removed, and cells lysed with 0.1 M HCl/1% Triton X-100 at RT. Cell lysates were centrifuged and supernatants were assessed for LT-induced cAMP or ST-induced cGMP by using direct cAMP/cGMP ELISA kits  according to manufacturer’s instructions. LT neutralization activity was calculated by multiplying the plasma/serum dilution factor with the percentage of LT neutralization at the corresponding plasma/serum dilution used. Neutralizing capacity of anti-LTB or anti-CTB antibodies in serum was assessed in GM1 binding assay. Serum dilutions were incubated with 0.5 ng LTB or CTB (Sigma-Aldrich), and the amount of LTB or CTB that remained free from antibody binding was quantified by binding to GM1-coated plates by ELISA using an anti-cholera toxin beta antibody (Sigma-Aldrich) for detection. LT or ST neutralization was assessed by blocking of LT-induced cAMP or ST-induced cGMP induction in T84 human colon epithelial cells as previously described ,12. BrieIgG antibody responses to LTB peptides were assayed using peptide array analysis. Medium-density arrays were created using laser jet-assisted on-chip synthesis technology. On these array chips, 93 12-amino acid (12-mer) overlapping LTA peptides and 74 12-mer LTB peptides were spotted onto each chip; the peptide location on the chip was randomized to reduce the influence of any spatial bias of the chip. Peptide sequences were of the LT1 variant , and also included peptide sequence variants of both LTA and LTB from LT2, LT3, LT4, and LT6 variants . The peptide sequences selected were sequential and overlapping and spanned the entire amino acid sequences of LTA and LTB with a sequence overlap of 10 amino acids between each peptide. In addition to LTA and LTB peptides, control peptides were also included on the array . All peptides were printed on 16 separate sectors of each array chip, allowing analysis of 16 different samples on each array. To map antibody binding to each peptide, each serum sample was incubated in a 1/500-dilution to a peptide array sector containing all LTA and LTB peptides and control peptides, followed by washing and subsequent incubation by DyLight680-conjugated goat anti-human-IgG (Fc) antibody. Finally, fluorescence image scanning using a Innopsys InnoScan 710-IR microarray scanner, and subsequent digital image analysis was performed to detect antibody binding to each of the peptides on the chip. Chip printing and antibody analysis was performed by PEPperPRINT . Data for antibody binding to each peptide was generated by subtracting the assay background  from the foreground signal , for each sample. The cutoff for a positive signal was calculated as being 3 standard deviations above the average log fluorescence signal of the negative control peptide spots; this cutoff was 480 for IgG-responses.The study was conducted between January 2020 and February 2021 in Bangladesh in Mirpur, located in the Dhaka Metropolitan area. This urban research area has been subjected to field studies since 1987 and is densely populated with approximately 3.5 million individuals. Approximately 98% of the residents have access to tap water supplied by the municipality while the remainder use wells, hand pumps, and other sources such as ponds and rivers in the study area. As part of this study, participants were recruited from selected local health facilities in Mirpur as well as the icddr,b Dhaka hospital, presenting with clinical symptoms of diarrhea. A total of 1097 diarrheal patients  presenting with watery stool were screened for the type of infection by culture and PCR of the stool samples. One hundred-thirty ETEC-infected patients were identified and 97 met the inclusion/exclusion criteria and were enrolled in the study. Among the enrolled ETEC-positive adult patients, 43 completed the follow-up visits and seven were lost to the follow-up. In the pediatric group, 46 completed the follow-up visits and one was lost to the follow-up. The age and gender distribution of enrolled participants are detailed in The ETEC infection was typed in the enrolled patients regarding the presence of the LT and ST genes . Of the The plasma from all the enrolled ETEC-infected patients was assessed for LTB-reactive IgG and IgA antibodies in acute, early, and late convalescence phases . The individual titers are shown in There was no correlation between the IgG and IgA antibody titers against LTB in adults, while in children a strong correlation was observed after infection with an LT+ ETEC strain . In addition to the LTB responses, we assessed the anti-LTA IgG antibody responses in all the ETEC infected adults and children enrolled in the study . Notably, only a few individuals had high reactivity against the 12-mer peptides and even fewer samples showed reactivity to overlapping peptides . In thesImportantly, the neutralizing potency of the plasma samples did not correlate with the IgG antibody responses to linear epitopes.The plasma samples from all the enrolled ETEC-infected patients were assessed for IgG antibodies against ST at acute, early, and late convalescence phases  . Of the Plasma from these four subjects was assessed in an ST neutralization assay based on the ST-induced cGMP induction in T84 human colon epithelial cells. No significant increase in the ST-neutralizing capacity of plasma collected at day 7 or 30 compared to day 2 plasma of the same subject could be detected in any of the analyzed samples. This suggests that the anti-ST IgG antibodies present in the plasma after natural ST+ ETEC infection were not sufficient to achieve neutralization of ST under the experimental conditions tested.The plasma from 40 healthy adults from Bangladesh was also collected within this study. These samples were compared for the anti-ST IgG to those from an endemic sample collection of 401 healthy adults and a non-endemic collection of 49 sera . While tETEC infection is an important cause of diarrhea in children in low- and middle-income countries (LMIC) and is also one of the main causes of traveler’s diarrhea (TD). The vaccine development efforts against ETEC have been mainly focused on the B-subunit of the heat-labile toxin (LTB) and surface colonization factors .In the study reported here, we found that 42% of the enrolled children were infected with an LT-only containing ETEC strain. In contrast, only 14% of the adults were infected with an ETEC strain that expressed only LT (LT+ST−). It can be speculated that adults have gained protective immunity against LT through multiple previous ETEC infections, hence less adults were getting sick from LT-only-expressing ETEC or producing milder symptoms which did not need to be treated in a medical center. In contrast, ST, being a poor antigen, did not induce sufficient protective immunity, leaving even adults vulnerable to ST-expressing ETEC. This is supported by data from multiple studies. The contribution of LT+ or ST+ ETEC to diarrheal stools has been shown to be of a similar proportion in children in their first and second year of life . AnotherIt has been previously shown that LT+ ETEC infection conferred a 45% protection against symptomatic infection with LT+ ETEC . In thisWe found that both LT subunits were immunogenic and contributed to the neutralization activity. Interestingly, the anti-LTB IgG and IgA responses correlated in the children but not in the adults where we detected higher IgA levels even when the IgG levels were low.The anti-LTB IgG titers strongly correlated with the neutralizing potency in children and adults. This confirms results from oral vaccination with the LTB-containing vaccine ShigETEC, for which LTB IgG titer also correlated with neutralizing potential . Due to The linear epitope mapping identified, for the most part, relatively weak antibody responses to LTB, which was inconsistent across different individuals. The epitope responses to LTA were stronger, and there were two particular peptides (positions 54–66 and 138–150) that elicited a response in almost all adults and children. These two peptides also overlapped with previously reported epitopes [In this study we found weak responses against ST after ST+ ETEC infection. While this could be due to the relatively small number of individuals included in the study, in general, the background level of anti-ST antibodies in endemic and nonendemic populations were not substantially different. Only a few plasma samples were identified with increased anti-ST IgG levels, but none were high enough to show neutralizing activity. This is supported by a previous observation that natural ETEC infections do not appear to induce a meaningful immune response against ST, which is presumably due to the small size of ST . The pooThe improvement of the current vaccine development efforts to include large amounts of ST protein could provide a tool to tackle the important contribution of ST-containing ETEC strains to severe disease and death among children.


Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user’s camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 20 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns. Neurodegenerative disorders like Parkinson’s disease (PD) are an increasingly significant health crisis in part due to aging populations. PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra region of the brain . Decadesin vivo. In addition, these neurons do not form their natural networks and the role of their dysfunction cannot be contextualized to an organismal scale  strain was utilized. The 6-OHDA exposures used RB1600 [tub-1(ok192)], and RB2237 [tub-2(ok3024)], which were crossed with BY200. Worms were cultured on K-agar ) or LIU2 (ldrIs2 [mdt-28p::mdt-28::mCherry + unc-76(+)]) were independently crossed with (PNG)Click here for additional data file.S6 Fig.(PNG)Click here for additional data file.S7 FigA. An example of an image that would not be accurately analyzed by AUDDIT. The frame of the image contains multiple worms. In addition, the head region of one of the worms is not fully in the frame. The head region of the other worm is oriented such that two of the four CEP dendrites are overlapping, making it impossible to analyze each of them. B. AUDDIT would be able to accurately analyze the CEP dendrites of this worm. The head region of the worm is centered in the image, and there are no other worms or objects around the edges of the frame. The worm is oriented such that all four CEP dendrites are visible, and thus can each be analyzed.(PNG)Click here for additional data file.


What is the effect of an artificial intelligence–based smartphone app vs usual care alone or web-based self-management support on musculoskeletal health in patients with neck and/or low back pain referred to specialist care?In this randomized clinical trial of 294 adults with neck and/or low back pain referred to specialist care, the individually tailored app adjunct to usual care did not significantly improve musculoskeletal health more than usual care alone or nontailored web-based support at 3 months.These findings suggest that further research is needed to investigate the utility of implementing digitally supported self-management interventions for those with neck and/or low back pain referred to specialist care and to identify instruments that capture changes in self-management behavior. Self-management is a key element in the care of persistent neck and low back pain. Individually tailored self-management support delivered via a smartphone app in a specialist care setting has not been tested.To determine the effect of individually tailored self-management support delivered via an artificial intelligence–based app (SELFBACK) adjunct to usual care vs usual care alone or nontailored web-based self-management support (e-Help) on musculoskeletal health.This randomized clinical trial recruited adults 18 years or older with neck and/or low back pain who had been referred to and accepted on a waiting list for specialist care at a multidisciplinary hospital outpatient clinic for back, neck, and shoulder rehabilitation. Participants were enrolled from July 9, 2020, to April 29, 2021. Of 377 patients assessed for eligibility, 76 did not complete the baseline questionnaire, and 7 did not meet the eligibility criteria ; the remaining 294 patients were included in the study and randomized to 3 parallel groups, with follow-up of 6 months.Participants were randomly assigned to receive app-based individually tailored self-management support in addition to usual care (app group), web-based nontailored self-management support in addition to usual care (e-Help group), or usual care alone .The primary outcome was change in musculoskeletal health measured by the Musculoskeletal Health Questionnaire (MSK-HQ) at 3 months. Secondary outcomes included change in musculoskeletal health measured by the MSK-HQ at 6 weeks and 6 months and pain-related disability, pain intensity, pain-related cognition, and health-related quality of life at 6 weeks, 3 months, and 6 months.P = .60). The adjusted mean difference between the app and e-Help groups was 1.08 points .Among 294 participants , 99 were randomized to the app group, 98 to the e-Help group, and 97 to the usual care group. At 3 months, 243 participants (82.7%) had complete data on the primary outcome. In the intention-to-treat analysis at 3 months, the adjusted mean difference in MSK-HQ score between the app and usual care groups was 0.62 points (95% CI, −1.66 to 2.90 points; In this randomized clinical trial, individually tailored self-management support delivered via an artificial intelligence–based app adjunct to usual care was not significantly more effective in improving musculoskeletal health than usual care alone or web-based nontailored self-management support in patients with neck and/or low back pain referred to specialist care. Further research is needed to investigate the utility of implementing digitally supported self-management interventions in the specialist care setting and to identify instruments that capture changes in self-management behavior.NCT04463043ClinicalTrials.gov Identifier:  This randomized clinical trial assesses the effect of an artificial intelligence–based self-management app vs usual care alone or web-based self-management support on musculoskeletal health in adults with neck and/or low back pain referred to specialist care. However, knowledge of their applicability and effects in the specialist care setting is lacking. Specialist care is characterized by high service demands and limited capacity, which can result in long waiting times for first consultations; thus, implementing evidence-based self-management digital tools at the time of referral might improve quality of care and patient outcomes in this setting.Current best evidence suggests that self-management for persistent pain should be tailored to the patient’s individual needs and capabilities.16 recently evaluated the effectiveness of a knowledge-based artificial intelligence (AI) decision support system entitled SELFBACK, which supports individually tailored and evidence-based self-management of low back pain via a smartphone app. Results of a randomized clinical trial (RCT)17 indicated a small but favorable effect of the app-based intervention compared with usual care on pain-related disability among patients receiving primary care.18 For the current RCT, we adapted the content of the app to also target patients with neck pain.19 In addition, we developed a web-based self-management intervention without individual tailoring (e-Help)19 to serve as an active control condition.A previous studyThe primary aim of this RCT was to determine the effect of the app adjunct to usual care vs usual care alone on musculoskeletal health in patients with neck and/or low back pain referred to specialist care. The secondary aim was to determine the effect of the app on musculoskeletal health compared with the e-Help intervention.19 All participants provided written informed consent before entering the trial. The trial followed the Consolidated Standards of Reporting Trials (CONSORT) guideline for RCTs.20The trial was approved by the Norwegian Medicines Agency and the regional ethics committee in Norway. The trial protocol  was prerWe recruited adults 18 years or older with neck and/or low back pain who had been referred and accepted to the multidisciplinary outpatient clinic for back, neck, and shoulder rehabilitation at St. Olav’s Hospital in Trondheim, Norway. Inclusion criteria included owning a smartphone (iOS or Android operating system) with internet access and a working email address. Exclusion criteria included warning signs indicating possible cancer, fracture, cauda equina syndrome, infection, or other conditions prioritized for urgent treatment or examination; inability to take part in exercise or physical activity ; inability to speak and/or read the Norwegian language; or enrollment in the ongoing SELFBACK trial in the primary care setting.All patients with neck and/or low back pain who were referred to and accepted on a waiting list for the outpatient clinic between July 9, 2020, and April 29, 2021, were invited to participate. Administrative staff at the clinic identified potential participants based on the information available at referral. Potential participants were sent an invitation text message with a link to a registration form containing information about the trial, eligibility questions, and a digital consent form. Patients who consented and fulfilled the eligibility criteria were invited by email to complete the online baseline questionnaire. Those unable or unwilling to complete the baseline questionnaire were excluded from the study. Of 377 patients assessed for eligibility, 76 did not complete the baseline questionnaire, and 7 did not meet the eligibility criteria; the remaining 294 patients were included in the study.After completing the baseline questionnaire, participants were randomized to receive (1) the app-based intervention adjunct to usual care (app group), (2) the e-Help web-based intervention adjunct to usual care (e-Help group), or (3) usual care only . Randomization was performed using a web-based trial management system administered by the Unit of Applied Clinical Research at the Norwegian University of Science and Technology. Participants were randomized using permuted blocks with random sizes from 4 to 20 and 1:1:1 allocation. To ensure concealed allocation, the block sizes were not disclosed to study personnel, and the randomization code was automatically generated by the trial system and released after participants were enrolled in the trial. After randomization, participants were informed about their group allocation via a text message .Participants in all 3 groups were instructed to follow any diagnostic- or treatment-related pathways as indicated by health care practitioners they may have consulted during the study period. At the first consultation at the clinic , all patients received usual care, which comprised a clinical examination and the offer of suitable treatment in accordance with current evidence-based guidelines. Treatment options included no further treatment, adjusted recommendations for primary care treatment, outpatient multimodal rehabilitation , or referral for surgical treatment.Participants randomized to the app group were instructed to install the app via a link provided in the text message containing the information about group allocation. Correspondingly, participants randomized to the e-Help group received a link to the website and login instructions . Participants randomized to the intervention groups had unrestricted access to the app or e-Help website throughout the study period.21The app provides participants with weekly and individually tailored self-management recommendations for physical activity, strength and flexibility exercises, and daily educational messages as well as access to different tools and resources  that participants could use at their convenience  along with the latest questionnaire information. By following the weekly recommendations, patients could collect badges and rewards displayed in the app. Moreover, push notifications triggered by the patient’s self-management behavior  were sent via the app to motivate and reinforce the desired self-management behavior.Case-based reasoning is a knowledge-based AI methodThe e-Help website offered evidence-based support and advice on self-management of neck and low back pain. Participants could access the website via their own device  at any time throughout the study period. The website has 4 main sections: (1) a home page, which describes the purpose of the e-Help intervention and how to use the e-Help resources; (2) educational messages; (3) videos and descriptions of strength and flexibility exercises; and (4) additional resources and educational tools, including instructions on goal setting, pacing techniques, sleep hygiene, and mindfulness along with practice audio files and links to relevant external websites. The content in sections 2 to 4 imitates the content in the app ; however, individual tailoring is not provided on the e-Help website. Clinicians at the outpatient clinic  and a patient representative were involved in the development of the website to ensure it aligned with treatment principles at the clinic.24 (MSK-HQ) scores between groups at 3 months. The Norwegian version of the MSK-HQ is a reliable and valid measure of musculoskeletal health among people on sick leave due to a musculoskeletal disorder.25 The MSK-HQ scale ranges from 0 to 56 points, with higher scores indicating better musculoskeletal health. In addition, we examined the difference in the proportion of patients reporting a 4-point or higher improvement in MSK-HQ score.Sociodemographic information was collected at baseline. Outcomes were assessed using an online questionnaire at baseline, 6 weeks, 3 months, and 6 months. The primary outcome was the mean difference in Musculoskeletal Health Questionnaire26; neck pain–related disability assessed by the Neck Disability Index 27; mean and worst pain intensity in the preceding week rated on a numerical rating scale 28; confidence in ability to cope despite pain assessed by the Pain Self-Efficacy Questionnaire 29; cognitive and emotional perceptions of illness assessed by the Brief Illness Perception Questionnaire 30; health-related quality of life assessed by the EuroQol 5-dimension questionnaire, weighted according to the Danish value set31  and the EuroQol visual analog scale 32; and overall improvement assessed by the Global Perceived Effect scale .33 Additional exploratory outcomes were also evaluated as prespecified in the statistical analysis plan.34Secondary outcomes included low back pain–related disability assessed by the Roland-Morris Disability Questionnaire Adverse events were defined as events causing participants to consult a health care professional due to circumstances potentially related to the interventions. Occurrences of adverse events were registered with the Norwegian health authorities when participants reported them to study personnel.19The planned sample size of at least 279 participants (93 in each group) was based on a power of 90% to detect a 4-point mean group difference in MSK-HQ score at 3 months, assuming an SD of 10 points, a correlation of 0.4 between repeated measures in the same participants, 2-sided α = .05, and a 30% study withdrawal rate during follow-up.35 In this analysis, baseline and all follow-up values were modeled as dependent variables, and mean baseline values were constrained to be equal in the 3 groups, thereby accounting for any random differences in the outcome variable at baseline. To account for the dependency in observations within participants over time, the linear mixed model included a random intercept for each participant.The primary intention-to-treat analysis estimated mean group differences (with 95% CIs) in MSK-HQ score at 3 months using a constrained longitudinal data analysis approach fit with a linear mixed model.34 The per-protocol analysis included 2 approaches. In the first approach, adherence was defined as having accessed the intervention at least once (for both intervention groups); in the second approach, adherence was defined as having generated 6 or more self-management plans during the first 12 weeks after randomization (for the app group only). Assumptions related to the normality and homogeneity of residuals and the normality of random intercepts were assessed for all models. Analysis of mean group difference at 6 months and analyses of secondary and exploratory outcomes followed the same analytic approach used for the primary analysis.All effect estimates were adjusted for potentially important estimators of the outcome, including age (years), sex , educational level , and baseline mean pain intensity in the past week . Preplanned sensitivity analyses of the primary outcome included (1) multiple imputation of missing values using a multivariate normal approach and 20 imputed data sets, (2) complete case analysis including participants with data for all time points, and (3) per-protocol analysis of participants who adhered to the intervention.We estimated the risk ratio for a 4-point or greater improvement in MSK-HQ score from baseline to 3-month follow-up using a Poisson generalized estimated equation (GEE) model. Similar GEE analyses were performed to estimate odds ratios for secondary binary outcomes using a logistic model. All GEE models assumed an exchangeable correlation structure with a robust variance estimator.P < .05. Analyses were performed by a blinded researcher (T.I.L.N.) using Stata software, version 17 (StataCorp LLC),36 and the interpretation of blinded results was published.37All estimates of precision were based on 2-sided tests. Statistical significance was defined as 2-tailed The flow of participants through the trial is shown in the P = .60) higher in the app group compared with the usual care group and 1.08 points  higher in the app group compared with the e-Help group  for the usual care group, 5.4 points  for the app group, and 4.1 points  for the e-Help group. There were no statistically significant differences in MSK-HQ scores among participants in the app group compared with other groups. The adjusted mean MSK-HQ score at 3 months was 0.62 points  in favor of the app group compared with the usual care group and 1.23  in favor of the app group compared with the e-Help group, although these results were not statistically significant (The percentage of participants reporting an improvement of 4 points or greater on the MSK-HQ from baseline to 3 months was 44.2% (38 of 86 participants) in the usual care group, 59.0% (46 of 78 participants) in the app group, and 46.8% (37 of 79 participants) in the e-Help intervention group. These proportions corresponded to adjusted risk ratios for improvement of 1.35   . At 6 mo = .004) . ExploraOne participant randomized to the app-based intervention reported hospitalization due to dizziness during participation in the trial. However, this event was deemed unlikely to be related to the intervention and was spontaneously resolved.This RCT found that among patients referred and admitted to specialist care due to neck and/or low back pain, the receipt of evidence-based and individually tailored self-management support via an AI-based app adjunct to usual care did not result in significantly improved musculoskeletal health at 3 months compared with usual care alone. The results for the primary and secondary outcomes were similar at 6 months.38 In this way, digital interventions can potentially be used to reinforce and support patients’ self-management. The results of this trial indicated that the app was not an effective treatment adjunct to usual care for patients with neck and/or low back pain referred to specialist care. We are not aware of other studies that have tested similar interventions for self-management in the specialist care setting. A previous RCT in the primary care setting17 found a small but favorable reduction in pain-related disability among people with low back pain receiving the app-based intervention adjunct to usual care compared with those receiving usual care alone. Several factors related to the trial designs may partly explain these contrasting results. For example, unlike the current trial, clinicians in the primary care trial17 were involved in the recruitment of participants. Some evidence suggests that endorsement by a clinician may be important to facilitate and reinforce patients’ engagement in self-management.39 The different onboarding procedure used  might also have influenced participant engagement with the app-based intervention, which was lower in the current trial. In addition, patients referred to specialist care might represent a group with more complex symptom profiles compared with those receiving primary care,40 which could potentially influence their ability to self-manage. The process evaluation conducted along with the trial will provide more detailed insight into barriers and facilitators related to the uptake and use of digital interventions in specialist care.19Multidisciplinary pain treatment in specialist care commonly includes elements intended to improve patients’ self-management skills.This study has several strengths. These strengths include the 3-group design ensuring an active comparison group; well-balanced baseline characteristics between groups, suggesting successful randomization; a high retention rate at the primary end point of 3 months; and a preplanned blinded analysis.41 we used musculoskeletal health as a proxy measure. This measure might have been suboptimal for detecting changes in self-management behavior if they existed.The study also has limitations. First, the uptake of the interventions was suboptimal . Consistent with the pragmatic nature of the trial, participants were required to install the app themselves, but phone assistance was offered to those who needed it. This process might have influenced uptake of the app, particularly among participants with limited digital literacy. Low engagement with the intervention could explain the lack of effect on the primary outcome, even if the app itself was potentially effective. However, our per-protocol analyses did not support this notion. In addition, given the lack of consensus on outcome measures for self-management behavior,In this RCT of patients with neck and/or low back pain referred to specialist care, individually tailored and evidence-based self-management support delivered via an AI-based app adjunct to usual care did not significantly improve musculoskeletal health at 3 months compared with usual care alone or web-based self-management support without individual tailoring. Future studies should investigate the utility of implementing digitally supported self-management interventions in the specialist care setting and identify instruments that capture changes in self-management behavior.


Fetal distress is a symptom of fetal intrauterine hypoxia, which is seriously harmful to both the fetus and the pregnant woman. The current primary clinical tool for the assessment of fetal distress is Cardiotocography (CTG). Due to subjective variability, physicians often interpret CTG results inconsistently, hence the need to develop an auxiliary diagnostic system for fetal distress. Although the deep learning-based fetal distress-assisted diagnosis model has a high classification accuracy, the model not only has a large number of parameters but also requires a large number of computational resources, which is difficult to deploy to practical end-use scenarios. Therefore, this paper proposes a lightweight fetal distress-assisted diagnosis network, LW-FHRNet, based on a cross-channel interactive attention mechanism. The wavelet packet decomposition technique is used to convert the one-dimensional fetal heart rate (FHR) signal into a two-dimensional wavelet packet coefficient matrix map as the network input layer to fully obtain the feature information of the FHR signal. With ShuffleNet-v2 as the core, a local cross-channel interactive attention mechanism is introduced to enhance the model’s ability to extract features and achieve effective fusion of multichannel features without dimensionality reduction. In this paper, the publicly available database CTU-UHB is used for the network performance evaluation. LW-FHRNet achieves 95.24% accuracy, which meets or exceeds the classification results of deep learning-based models. Additionally, the number of model parameters is reduced many times compared with the deep learning model, and the size of the model parameters is only 0.33 M. The results show that the lightweight model proposed in this paper can effectively aid in fetal distress diagnosis. Fetal distress is a syndrome of respiratory and circulatory insufficiency caused by intrauterine fetal hypoxia during labor and is closely associated with changes in fetal heart rate signals . Fetal dWith the development of artificial intelligence technology, scholars worldwide are committed to developing fetal health-assisted diagnosis systems based on machine learning and deep learning to help healthcare professionals analyze CTG signals objectively and correctly. Lightweight models and miniaturization have become a trend in many application scenarios, so an increasing number of academics are focusing on lightweight network models that can be deployed and run directly on mobile devices. The MobileNet series  and Shuf(1) The matrix feature map based on wavelet packet coefficients is constructed to refine the FHR signal in multiple frequency bands and used as input to the model. Different wavelet basis functions are selected to generate multiple feature maps to vote on the sample classification results.(2) The cross-channel interactive attention module is embedded in the tail of the ShuffleNet-V2 base unit to generate an ECA-Shuffle unit to achieve effective multichannel feature fusion without dimensionality reduction.(3) A lightweight fetal distress-assisted diagnosis network based on the FHR signal, LW-FHRNet, is proposed. Conventional convolution with ECA-Shuffle units ensures effective channel feature fusion while reducing model complexity and enhances the model’s ability to classify fetal distress.Aiming at the complexity and considerable computation in existing deep learning-based fetal distress algorithm models, this paper introduces a lightweight network architecture to design a lightweight fetal distress-assisted diagnosis network based on FHR. Additionally, to further improve the feature extraction ability and classification effect of the network, the attention mechanism is incorporated into the lightweight network to build a lightweight network unit (ECA-Shuffle) based on the cross-channel interactive attention mechanism. The main contributions of this paper are as follows.The rest of the paper is presented below. The architecture of the lightweight fetal distress-assisted diagnosis model based on the cross-channel interactive attention mechanism designed in this paper is shown in (1) If the FHR value is equal to 0 and the duration is greater than 15 s, the segment is removed directly; otherwise, it is linearly interpolated.(2) If the FHR value is unstable, i.e., the absolute value of two adjacent points is greater than 25 bpm, and interpolation is performed between the starting sampling point and the first point of the next stabilization segment. A stable segment is defined as five consecutive FHR values where the difference is less than 10 bpm.(3) If the FHR value is greater than 200 bpm or less than 50 bpm, it is filled in with Hermite spline interpolation.Clinically, the FHR signal is acquired mainly by an ultrasound Doppler probe placed in the abdomen of the pregnant woman. During the acquisition process, the signal is inevitably subject to a variety of noise interferences, such as the movement of the fetus and the pregnant woman, improper placement of the sensor and other external factors. The noise of the FHR is represented by spikes  and missing values  . AccordiNoise and missing value segments in the FHR signal can be effectively filtered out by the above interpolation method. In conjunction with the time requirement of clinical prenatal examination, this paper uses 20-min data segments for analysis. The preprocessed data are segmented into 20-min time segments to obtain multicomponent segment data. The waveform obtained using the above preprocessing method is shown in As a non-stationary and non-linear time series, FHR contains complex physiological and pathological information. Wavelet packet decomposition is a discrete analysis method of non-stationary signals that can select the appropriate spectral band according to the signal characteristics and improve the time-frequency analysis resolution . In thisnth layer decomposition, the wavelet packet transform provides 2n different subspaces, and each subspace corresponds to a frequency band.jth subfrequency band at the ith layer decomposition, ith layer decomposition Wavelet packet decomposition can be implemented using a series of convolutions with high-pass filters and low-pass filters. The high-pass filter To increase the number of datasets to obtain better model effects, db1∼db5 wavelet basis functions are selected for wavelet packet coefficient decomposition in this paper. Therefore, five wavelet packet coefficient matrix maps can be obtained for each data segment to enhance the dataset. Meanwhile, each wavelet packet matrix coefficient map is resized to 224*224*3 pixels as the input layer of the neural network model. The feature map construction based on wavelet packet coefficients is shown in To meet the application of deep neural networks on embedded and mobile terminals and maintain excellent performance, lightweight network models have emerged. In particular, the lightweight models of the MobileNet series and the ShuffleNet series are the most widely used. Depthwise separable convolution, pointwise convolution, group convolution, channel shuffle and channel separation are used to reduce the number of model parameters and speed up the model computation time.Recently, the channel attention mechanism has been shown to have great potential in improving the performance of deep convolutional neural networks. By assigning different weights to each part of the input, more important information can be extracted to help the model make more accurate judgments without imposing greater overhead on the model’s computation and storage.Inspired by the above work, a lightweight network based on a cross-channel attention mechanism, LW-FHRNet, is proposed in this work to assist in the diagnosis of fetal distress symptoms, as shown in via concat, and the feature matrix depth is kept constant. The ECA strategy is also used at the end of the structure.Based on the ShuffleNet-V2 units, this study constructs two types of ECA-Shuffle units by integrating the cross-channel attention module without dimensionality reduction, as shown in The lower half of the ECA-Shuffle unit is the cross-channel interactive attention module without dimensionality reduction. The detailed structure is shown in ω=σ(Wy)If the wConsidering each channel and its k nearest neighbors, computing local cross-channel interaction information instead of all channels effectively improves computational efficiency. This efficient channel attention calculation can be quickly implemented by 1D convolution. Thus, To avoid resource-consuming cross-validation adjustment, an adaptive method is used to select the appropriate q. C=ϕkSince thThe details of the lightweight network: LW-FHRNet structure designed in this work are shown in The process of the fetal distress classification algorithm based on a lightweight network is described in The database in this paper uses the publicly available dataset CTU-UHB, which comes from the Czech Technical University in Prague (CTU) and the University Hospital in Brno (UHB) . A totalThe pH value is a marker of blood acid-base balance and can provide information on possible fetal acidosis caused by intrauterine hypoxia. A lower pH value represents a more severe degree of fetal acidosis . showed In this paper, each 20-min segment of FHR data is subjected to wavelet packet decomposition based on db1 to db5 wavelet basis functions to obtain five wavelet coefficient matrix feature maps. Category attribution is determined by voting on the 5 feature maps. The category voting process is shown in The network structure proposed in this paper is trained and tested on the CTU-UHB dataset. The experimental platform is a computer equipped with an Intel Xeon(R) CPU E3-1535M v6 @ 3.10 GHz x 8, Quadro P5000 GPU and 32 G RAM. The system is Ubuntu 18.04.6LTS, the development environment is TensorFlow 2.6.2, and the language used is Python.To evaluate the classification performance of the model, accuracy, precision, recall and F1-Score metrics are used in this paper. Additionally, model parameters and model size are introduced to evaluate the complexity of lightweight models. Finally, sensitivity (Se) and specificity (Sp) are used to observe the discriminatory ability of the model between abnormal and normal samples.The commonly used lightweight networks MobileNetV3-Small, MobileNetV3-Large and ShuffleNet-V2 are introduced as the baselines of this research. MobileNetV3 introduces the channel attention module based on MobileNetV2 to enhance the adaptive capability of the model by assigning different weights to different channels. MobileNetV3 has two versions: small and large. ShuffleNet-V2 proposes the concept of channel separation to replace group convolution to further improve the inference speed.ith layer is decomposed to obtain 2i frequency bands. The 2D image is constructed according to the frequency from the highest to the lowest. The frequency range of the jth frequency band is Wavelet packet decomposition with different numbers of layers can obtain different detailed information. The sampling frequency of the raw data is 4 Hz. The The channel attention mechanism has great potential to improve the performance of deep convolutional neural networks. In this paper, we introduce a cross-channel local interaction attention strategy without dimensionality reduction to improve the performance of lightweight models. Experiments are conducted on the dataset of this paper using a lightweight network with and without an ECA module. The confusion matrix of whether the proposed lightweight model contains ECA modules is shown in To clarify the performance of the network, this paper performs a comparative test with different lightweight networks. The classification performance of fetal distress under different lightweight networks is measured using accuracy, precision, recall, F1-score and model size metrics. The test performance comparison of the LW-FHRNet network with other commonly used lightweight networks is shown in LW-FHRNet incorporates an efficient cross-channel attention mechanism without downscaling on the base unit of ShuffleNet-V2. The channel interaction strategy effectively improves the performance of channel attention and enables LW-FHRNet to have a more accurate recognition performance. The ROC curves of LW-FHRNet and other commonly used lightweight network models are shown in In this paper, a lightweight network based on cross-channel interactive attention mechanism is proposed to effectively fuse channel features and reduce model complexity to help obstetricians to objectively assess fetal distress. In the experiments, the classification effects of wavelet packet decomposition with different layers as feature maps were first compared. And the optimal number of wavelet packet decomposition layers was chosen as 2-layer. Then two different network architectures (LW-FHRNet and LW-FHRNet-without-eca) were used. The results showed that the attention machine module effectively improves the classification performance of fetal distress. Finally, a comparison with other lightweight models was made to show that the lightweight network proposed in this paper outperforms other common lightweight networks.To analyze the significance of the results, the algorithm in this paper is compared with recent related work in the diagnosis of fetal distress using the CTU-UHB database. The results are shown in In conclusion, the lightweight network model based on the cross-channel interactive attention mechanism proposed in this paper achieves better classification results in fetal distress diagnosis. The ShuffleNet-V2 unit combined with the local cross-channel interactive attention mechanism is used to build a lightweight network, which ensures a low number of parameters and achieves effective network performance improvement.However, one limitation of the study in this paper is the criteria for discriminating between normal and distressed samples. The current work generally endorses the use of umbilical artery blood pH as a criterion for classification, since pH is an objective response to the fetal oxygen cell supply  and alsoIn this work, a lightweight network (LW-FHRNet) based on ECA-Shuffle units is proposed for fetal distress classification of FHR signals. After preprocessing, the FHR signal is segmented into 20-min segments, and the wavelet packet decomposition operation based on db1 to db5 wavelet basis functions is performed on each segment. Each segment obtains five wavelet packet coefficient matrix feature maps, which are used as input to the model and vote on the classification result. The ECA-Shuffle unit performs feature extraction on the feature map to fully learn the feature information. We integrate an efficient local cross-channel interactive attention mechanism without dimensionality reduction to reduce model complexity and ensure performance improvement. In this paper, the CTU-UHB open source database is used to test the classification performance of the proposed network. A pH value of 7.05 was used as the gold standard for classification. The proposed algorithmic model achieves excellent results of 95.24%, 90.48%, and 100% for Acc, Se and Sp, respectively.Although the proposed lightweight network achieved good results in classifying fetal distress, there is still a gap to reach the clinical diagnosis level of physicians. In order to achieve better auxiliary diagnosis, we will do further exploration in future work. On the one hand, the data from clinical fetal heart monitoring contain simultaneous UC signals and FHR signals, but only FHR signals are used to assess fetal distress because of the poor quality of UC signals in publicly available datasets. In the clinic, the UC signal is also an important basis for physicians to diagnose fetal distress. Therefore, the combination of FHR signals and UC signals needs to be considered in further studies. On the other hand, we are considering more time-frequency transform features to improve the classification performance for fetal distress, including Empirical Wavelet Transform, Hilbert-Huang Transform, Singular Spectrum Analysis, etc.


Punica granatum L.) is one of the fruit species with the oldest cultural history. There are many traits to determine the quality of pomegranate fruits. Among them, soft-seeded feature of pomegranate fruit is important trait for the market value of the fruit. For this reason, the demand for pomegranate varieties with soft seeds has been increasing, especially in recent years. In this study, molecular markers associated with seed hardness were developed to distinguish pomegranate cultivars with soft-seeded feature based on genomic DNA at the early stages of the pomegranate breeding process. For this purpose, pomegranate genotypes and/or cultivars from the population involved in reciprocal crosses of hard-seeded Ernar, medium-hard-seeded Hicaznar, and soft-seeded Fellahyemez cultivars were grouped as soft-seeded or hard-seeded. Further, leaf samples were collected from individuals belonging to each group. Then, the genomic DNA was isolated from each plant separately, and equal amount of genomic DNA from individuals with the similar seed hardness were mixed for bulked segregant analysis (BSA). The bulked genomic DNAs of opposite characters were analyzed by polymerase chain reaction (PCR) using random decamer primers to develop random amplified polymorphic DNA (RAPD) markers associated with soft-seeded or hard-seeded pomegranates. A total of three RAPD markers were determined to distinguish the individuals having soft- or hard-seeded pomegranate genotypes and/or cultivars. As a result of the comparison of the DNA sequences of these RAPD markers, insertion-deletions (inDels) primers were designed to developed and validate a PCR assay to distinguish the soft- and hard-seeded pomegranate genotypes/cultivars from each other. The molecular markers developed in this study will enable us to distinguish soft-seeded pomegranate types easily in a short time at the early stages of the pomegranate breeding programs.The pomegranate ( Pomegranate, one of the ancient fruit species, has been consumed both fresh and juiced for thousands of years. The demand for the production and consumption of pomegranate fruit has been increased in recent years due to its important bioactive properties such as antimicrobial , antiparThe edible part of the pomegranate fruit is called aril and constitutes 45–52% of the total fruit weight. Aril consists of a white, yellow, pink, and red fleshy, juicy part and the seeds inside consist of the embryo with a shell surrounding it ,7. It haIn recent years, the demand for pomegranate varieties with soft seeds has been increasing. They have been preferred by consumers for their ease of eating and chewing, and by the food industry for their ease of processing . On the Many studies were carried out to determine how pomegranate seeds gain the soft-seeded trait up to date ,15,16,17Molecular markers display genetic differences between individuals with opposite traits in the form of nucleotide sequences and have been developed and used to accelerate the breeding process in many crops. This procedure is called marker assisted selection (MAS). Determination of the characteristics of the fruits could be possible without waiting for an average of 5–10 years for bearing fruits in perennial plants using MAS. Due to this time-saving process, it is also possible to reduce the space, cost, and labor ,26. In oPomegranate genotypes were obtained from hybridization of different combinations of hard-seeded Ernar, medium-hard-seeded Hicaznar, and soft-seeded Fellahyemez cultivars in 1990 in Turkey. These crosses were made in order to develop new pomegranate varieties and phenological, morphological, and pomological characteristics of the population were determined by different researchers at different time periods ,28 at thGenomic DNAs were isolated from collected leaves of soft- and hard-seeded genotypes using CTAB DNA isolation protocol developed by Doyle and Doyle . The amo2, 0.2 mM dNTP, 10 µM RAPD primer or SSR primers, 1.25 U Taq polymerase , and 25–75 ng DNA. The PCR amplification was performed using MJ Mini PTC1148 and ICycler  thermocyclers. PCR reactions were performed in thin-walled PCR plates or tubes in two different thermocyclers programmed as an initial denaturation of 5 min at 94 °C, followed by 35–40 cycles of denaturation for 30 sec-1 min at 94 °C, primer annealing for 30 sec-1 min at 35–65 °C, and primer extension for 2–3 min at 72 °C followed by one cycle of at 72 °C for 10 min for final primer extension. After the amplification, RAPD-PCR products were separated on a 1.5–3% agarose gel in 1 × TAE buffer [50 × ] and photographed under UV light using Mini BIS-Pro  gel imaging system. On the other hand, PCR products of SSR were separated using Qsep 100 fragment analyzer system .The bulked DNA samples containing genomic DNAs of pomegranate genotypes with hard- and soft-seeded were analyzed by polymerase chain reaction (PCR) method using 260 random primers  and 24 different SSR primer pairs previously developed for pomegranate ,37,38,39Only the intense and clear bands consistently produced using genomic DNAs isolated at different time and different PCR machines were considered to be polymorphic and selected for further analysis. To confirm the consistency of a selected polymorphic bands, repetitive PCR reactions were performed using genomic DNAs of individuals constituting the bulked groups. Validation of polymorphic bands was performed using DNAs from the other members of progeny population, but not used in bulk analysis.Polymorphic DNA fragments associated with hard and soft seed traits were cut from agarose gel and purified using QIAquick Gel Extraction Kit . The purified DNA fragments were cloned into the pGEM-T easy vector using the T-A cloning method with pGEM-T easy PCR cloning system , as suggested by the manufacturer’s instructions to determine their sequences. Escherichia coli JM109 cells by heat shock transformation and screening of the resulting bacterial colonies was performed by colony PCR using M13 forward (F) and reverse (R) primers located outside of the T-A cloning site. Colonies showing positive result by colony PCR were grown in liquid LB medium with ampicillin, and purification of the plasmid DNA was performed using the Mini Prep Plasmid DNA isolation Kit , according to the manufacturer’s instructions. For the confirmation of the presence of additional DNA in the plasmid, the purified plasmids were digested using the EcoRI enzyme . Sequencing of plasmids containing polymorphic DNA fragments was carried out by bidirectional Sanger sequencing using universal primers, M13 F, and M13 R. Sequences of the obtained polymorphic DNA fragments were analyzed using the Vector NTI suite program . BLASTN and BLASTX applications of the National Center for Biotechnology Information (NCBI) were used to compare the sequences with the other sequences in GenBank at the nucleotide and protein level, respectively. Comparisons of the sequences with each other were performed using the BLAST-alignment module of the NCBI program. Based on the sequence comparisons, the InDel primers (forward 5′ GGCCCTCACATATTAAGTTCAC 3’ and reverse 5′ GTATCTTGAAAGTCAATGAGCC 3’) were designed. These InDel primers were used with 10 µM concentration in a 25-μL PCR reaction mixture as described in The clones were transferred to Phenotypical groupings of the selected population based on their seed hardness were re-evaluated for two years in row in this study, and new groups were formed. Bulked DNA samples representing opposite group of individuals’, hard-seeded, and soft-seeded were formed based on this new grouping. As a result of PCR analyzes using bulked DNA samples and SSR primer pairs, no polymorphic band or marker distinguishing hard- and soft-seeded individuals was determined. However, RAPD-PCR analysis using different random primers  produced 10 polymorphic bands indicating the differences between two bulked samples. To test the consistency of the obtained polymorphic bands as well as their ability to distinguish between hard- and soft-seeded individuals, genomic DNAs of individuals analyzed several times by RAPD-PCR using these primers. After repeated RAPD-PCR analyses, polymorphic bands were consistently obtained only from OPK-12 and OPAI-08 primers. Moreover, the validity of these polymorphic bands was then confirmed by PCR analyses of individual genomic DNAs constituting the bulks. After multiple PCR analyses, three polymorphic bands produced by these two different primers that were able to distinguish individuals with hard- and soft-seeded pomegranate genotypes were identified. Two of these polymorphic bands, about 550 and 600 bp, were obtained from RAPD-PCR amplification using the OPK-12 primer. These two polymorphic bands were very close to each other in size, but one band was only in soft-seeded individuals with a size of 550 bp, and the other band was only present in hard-seeded individuals with a size of 600 bp band. These markers are named as RAPDSS1 and RAPDHS1 markers, respectively . On the The OPK-12 primer produced polymorphic bands between hard- and soft-seeded individuals. PCR product of 550 bp amplified from a soft-seeded individual (16–99) and 600 bp amplified from a hard-seeded individual (17–21) by OPK-12 primer were excised from the gel and cloned to determine their DNA sequences. After cleaning the vector sequences, 571 and 608 bp clean sequence were obtained from the cloned PCR fragments of genotype 16–99 and 17–21, respectively. The comparison of the nucleotide sequences revealed that both fragments were aligned with each other and there was 33 bp deletion in soft-seeded genotype 16–99 .Punica granatum which was submitted the GenBank from sequencing the soft-seeded variety, ‘Tunisia’. In addition, the results of BLASTX analysis demonstrated that this protein was found in GenBank as protein number XP_031400272 and its function has not yet been determined. On the other hand, BLAST P analyzes demonstrated that orthologs of this protein has been functioned as ‘Calcium-dependent lipid-binding domain-containing protein’ in perennial trees, Melia azedarach (KAJ4722804.1), and Salix purpurea (KAJ6719349.1). Furthermore, based on the analysis in the Arabidopsis Information Resource (TAIR) database, it was determined that the ortholog of this gene is At1g04540 and its function is defined as ‘Calcium-dependent lipid binding (CaLB domain) protein’. Studies in Arabidopsis have also been demonstrated that this gene is expressed during mature pollen stage, germinated pollen stage, flowering stage, petal differentiation and expansion stage, and plant embryo globular stage during plant growth and development.BLASTN analysis showed that 550 bp DNA from the soft-seeded genotype 16–99 matched with uncharacterized cDNA (GenBankacession: XM_031544412.1) of The sequence analysis of the soft- and hard-seeded genotypes revealed 33 bp deletion in the soft-seeded genotype. This region was the source of polymorphisms detected by OPK-12 and the sequences were identical to the coding sequences in the pomegranate genome. Based on sequence information obtained from the cloned RAPD fragments of OPK-12, two primers were designed to differentiate hard- and soft-seeded genotypes to convert the RAPD marker into more reliable sequence based markers in this study . Since tThis InDel marker was first tested by PCR analyzes of the individual genomic DNAs forming the bulks. This PCR analysis clearly showed that while only 156 bp fragment was amplified from hard-seeded genotypes, a 123 bp fragment was amplified from soft-seeded genotypes along with the 156 bp DNA fragment in the population. Later, the ability of this InDel marker to distinguish between hard- and soft-seeded individuals was tested by PCR reactions using DNAs from the other individuals’ in the population were not included in the bulk. All these results showed that the primers designed based on sequences obtained from polymorphic RAPD fragments were consistently differentiate soft- and hard-seeded genotypes in the hybrid pomegranate population .Pomegranate varieties with soft-seeded feature are very few and only found in an area limited to a few ecological regions in the world . For exaDevelopment of new pomegranate varieties with desired characteristics is very difficult and takes a long period of time by conventional breeding methods. Marker assisted selection (MAS), which is among the new breeding techniques, reduce the amount of time during breeding of new fruit cultivars ,44. MAS Up to date, no molecular marker to be used directly in the breeding process of pomegranates has been identified. However, there are some studies conducted for the identification of molecular markers related with some traits including soft-seed feature. For example, in a study conducted by Sarkhosh et al., , a correIn recent years, especially after completion of the pomegranate genome, there have been a number of studies to find the differences between soft- and hard-seeded pomegranates at DNA, RNA, and protein level. In these studies, although some differences were determined at DNA, RNA, and protein levels between the compared genomes, no molecular marker that can be used in the breeding process of pomegranates has been determined yet ,21,22,23In plant breeding studies, the MAS technique is used to select individuals with desired characteristics in a short time and effectively. In this study, molecular markers associated with soft- and hard-seeded characteristics were developed for the first time to use of MAS technique in pomegranate breeding studies. Among these markers, three of them were RAPD markers and the product of one of these RAPD markers were sequenced and converted to the sequence-characterized InDel marker. All the markers developed in this study will enable pomegranate breeders to select the desired soft-seeded pomegranate genotypes easily without waiting 5–6 years for bearing fruit and will ensure that the breeding process is carried out effectively and quickly.


Rheumatoid arthritis (RA) is a chronic autoimmune multisystem inflammatory disease in which lung involvement is the most common extra-articular manifestation. Parenchymal lung involvement or interstitial lung disease (ILD) is a significant cause of morbidity and mortality and there is a paucity of evidence-based guidance on how to best treat RA-ILD. This review article aims to evaluate the evidence from cohort studies and best real word data from registries. Extensive discussion of the relative merits and drawbacks of glucocorticoids, various biologics, small molecules and anti-fibrotics is presented. The limited available guidelines in RA-ILD are also discussed and a rational treatment algorithm is offered. Rheumatoid arthritis (RA) is a chronic autoimmune systemic inflammatory disease affecting 0.5–1.0% of the global population . It is bThe recognised risk factors for RA-ILD development and poorer prognosis include age greater than 65, smoking, male gender, anti-citrullinated protein antibodies (ACPA), human leukocyte antigen HLA-DR4, a usual interstitial pneumonia pattern (UIP) of disease and subcutaneous nodules .Importantly, the histologic and high-resolution CT (HRCT) pattern of lung involvement has a significant impact on the prognosis and response to immunosuppressive treatment. The UIP pattern is seen in approximately half the cases of RA-ILD. The non-specific interstitial pneumonia (NSIP) pattern of disease is present in about a quarter of cases, followed then by the organising pneumonia (OP) pattern in about 10–15% of cases. The lymphoid interstitial pneumonia (LIP) pattern is seen rarely and tends to be treatment-resistant given that damage cannot be reversed on account of the cystic hollowed-out nature of parenchymal involvement. The natural history of the UIP pattern is similar to idiopathic pulmonary fibrosis and tends to be unrelenting if untreated. It is characterised by a HRCT appearance that is bilateral and predominantly basilar, with also subpleural fibrosis, cyst formation (honeycombing) and traction bronchiectasis. The NSIP pattern tends to be bilateral with peripheral ground-glass opacities, septal thickening and consolidation. Traction bronchiectasis may also be a feature; subpleural sparing is typical. The OP pattern features patchy but dense areas of lobar airspace consolidation on HRCT. There may be some ground-glass change surrounding these foci of consolidation. Lastly, the LIP pattern features thin-walled cysts with or without ground glass change ,9,10,11.As a general rule of thumb, the UIP and LIP patterns are considered irreversible, whereas the NSIP and OP patterns can resolve with glucocorticoids (GCs) and other immunosuppressive treatments. While damage cannot generally be reversed in UIP and LIP patterns, that does not mean there is no role for immunosuppressive treatment. On the contrary, the UIP pattern shows the stabilisation of both HRCT appearance and pulmonary function tests (PFTs) over time with certain treatments. This is a key distinction between the UIP pattern of disease seen in RA-ILD and the IPF-typical HRCT pattern of disease. While they have the same radiographic appearance, disease is responsive to both immunosuppression and anti-fibrotics in UIP RA-ILD, whereas anti-fibrotics are only able to slow deterioration in IPF. Both the NSIP and OP patterns of disease in RA-ILD respond very well to immunosuppression, showing the reversal or resolution of HRCT findings. The reason for this is that the bulk of the HRCT picture is likely inflammation in NSIP and OP, which can be reversed, whereas the typical findings on HRCT in UIP and LIP are reflective of fibrotic irreversible established changes. Taken from patients attending our centre, Despite the associated morbidity and mortality, there has been little consensus regarding guidelines for the treatment of RA-ILD patients. Unfortunately, there is little informed evidence by way of randomised control trials (RCTs) for potential treatments in RA-ILD. In terms of the hierarchy of evidence, most of the accepted evidence base comprises cohort, case–control, and cross-sectional studies. This review article aims to give a comprehensive review of these key studies so that practicing physicians are better informed of the possible treatment options.A systematic literature search for relevant articles was performed using PubMed, Embase and the Cochrane central register of controlled trials. The search was performed with no date limits and was last updated on 26 September 2023. The keywords ‘rheumatoid arthritis’ AND ‘interstitial lung disease’ OR ‘RA-ILD’ AND (‘treatment’ OR ‘therapeutics’) were used. The reference lists of key articles were also reviewed.While one class of RA therapeutic may have a comparable effect to another on synovial disease, the same is not necessarily true when it comes to pulmonary disease. An in-depth exploration of each drug class and their beneficial, or perhaps deleterious, effect on ILD stabilisation is provided. The evidence base dispelling the notion that methotrexate (MTX) exacerbates RA-ILD is highlighted and our preferred treatment strategy, which combines MTX with more modern agents, is offered. The relative merits of each therapeutic immunosuppressive agent are detailed in There remains much confusion regarding the role of GCs in the treatment of RA-ILD. The British Thoracic Society (BTS) produced the first guidelines for RA-ILD back in 2008. This recommended the use of prednisolone at 0.5 mg/kg/day for 1–3 months as the first-line treatment. It recommended a taper to less than 10 mg/day, with the addition thereafter of a disease-modifying anti-rheumatic drug (DMARD) . Of courOne case–control study evaluating the effect of oral GCs on RA-ILD mortality found that patients treated with prednisolone for more than 3 months had a greater mortality risk than those who did not receive prednisolone, with a relative risk (RR) of 2.06. The RR from respiratory causes was greater still at 2.75. The authors do note however that the preponderance of the usual interstitial pneumonia (UIP) pattern in the treatment group may explain the greater mortality seen . SimilarThe findings from Scott et al.’s study and the PANTHER trial contrast with the results of a retrospective case–control study performed by Song et al.; this study found that treatment with steroids, either as monotherapy or in combination with other immunosuppressants in 84 RA-ILD patients with a UIP pattern, improved or stabilised disease in 50% of the cohort. However, there was no statistically significant survival benefit compared with the untreated group despite the numerical trend . NeverthThe use of short-term steroids in combination with MTX and biological DMARDs (bDMARDs) may be reasonable on a case-by-case basis in NSIP, OP or LIP patterns of disease where the trajectory of progression is rapid and the patient cannot afford to wait for long-lasting immunosuppressants to take effect.MTX remains the anchor DMARD and the cornerstone of treatment in RA. Concern over a potential link between MTX and pulmonary toxicity first arose in 1983 and took many years to clarify . The keyp = 0.0006). In patients with RA-ILD, ILD detection was significantly delayed in the MTX ever-users compared to never-users  [p-value 0.04 by exact test). They also report that no cases could be considered probable or definite as too little information was provided [Further supportive evidence regarding the inverse relationship between MTX exposure and RA-ILD comes from Juge et al., as data from their derivation and validation samples showed a combined adjusted odds ratio (OR) of 0.43  . The fin< 0.001) . Lastly,< 0.001) . The CIRprovided . While pThere is a large body of evidence to support the idea that MTX is protective against RA-ILD development and progression. As general guidance, the use of MTX as a first-line treatment in RA-ILD is a reasonable evidence-based approach. However, those with a poor baseline pulmonary reserve, who are unlikely to tolerate MTX pneumonitis, should not be started on the drug for arthritis in the first instance.The controversy over the role of tumour necrosis factor inhibitors (TNFi) in RA has continued for the past 20 years. The pioneering RCTs of TNFi in RA were powered and designed to demonstrate efficacy in reducing the articular and synovial components of disease, not the stabilisation of RA-ILD or the effect on incident ILD. Consequently, the highest yield data comes from large biologic registries. Data from the British Society for Rheumatology Biologics Register (BSRBB) were analysed by Dixon et al. They report an overall prevalence of ILD of 2.6% in RA patients from the register. The prevalence of ILD was significantly higher in the TNFi-treated patients at 2.9% (299/9294) compared with 1.8% (68/2454) in the conventional DMARDs (csDMARD)-treated patients. Despite this, there was no significant difference between groups in terms of the standardised mortality or ILD mortality, although ILD mortality was numerically higher with TNFi treatment (15/70) than with csDMARD treatment (1/14) 21% vs. 7%) [% vs. 7% The BSRBB also provided some very insightful information on the relative efficacy of TNFi versus rituximab (RTX) in RA-ILD patients. Druce et al. compared the 5-year mortality rates in 43 RA-ILD patients treated with RTX as a first-line treatment with 309 RA-ILD patients treated with TNFi as a first-line treatment. Adjusting for confounders, RTX compared more favourably to TNFi, with a 5-year mortality hazard ratio (HR) of 0.53 . While sp = 0.024). Notably, of the 12 non-TNFi biologic patients, 9 were treated with tocilizumab (TCZ) and 3 with abatacept (ABA) [p < 0.05. Amongst the non-TNFi group, TZC- and ABA-treated patients did not differ in this regard. Of the 58 RA patients with pre-existing ILD, 14 progressed, all of which were in the TNFi group [The Japanese experience is similar. Nakashita et al. performed a prospective case–control study of 58 RA-ILD patients. The incidence of ILD exacerbation with TNFi versus non-TNFi biologics was statistically significantly higher, with 30.4% (14/46) in the former and 0% (0/12) in the latter (pt (ABA) . This stWhether or not TNFi treatment increases the risk of RA-ILD development remains not entirely clear. What is almost certain at this point is that non-TNFi bDMARDs such as ABA, RTX and perhaps TCZ are better treatments to use when attempting to arrest RA-ILD progression. As a result, it is the author’s opinion that the treatment should be optimised by switching from TNFi to ABA or RTX in any RA patient with evidence of clinically significant ILD or progression on TNFi.The bulk of the early evidence base for B-cell depletion and the use of RTX in RA-ILD comes from three cohort studies: two prospective and one retrospective. The first comparative cohort study in which RTX was compared with the TNFi class comes from the UK BSRBB. This prospective observational comparative study, conducted between 2008 and 2017, screened 1632 RTX and 15,644 TNFi patients. It compared mortality in patients with RA-ILD who received RTX or TNFi as their first biologic. Only 45 RTX patients and 309 TNFi patients met the RA-ILD inclusion criteria. As discussed earlier, the adjusted 5-year mortality in rituximab-treated patients is approximately half that of the TNFi-treated patients , but the difference was not statistically significant . NeverthThe second landmark UK paper investigating the role of rituximab in RA-ILD treatment was also published in 2017. This large single-centre retrospective observational cohort study screened 700 RA patients treated with RTX. In total, 56 patients with ILD were identified and 44 had available PFT data. The progression of ILD was defined as a decrease in pre-RTX (FVC) > 10% or a predicted decrease in the diffusion capacity of carbon monoxide (DLCO) > 15%. In total, 23/44 (52%) remained stable and 7/44 (16%) improved. In total, 14/44 (32%) progressed despite RTX treatment. In total, 9/56 (16%) of the cohort died during follow-up from progressive ILD. Unsurprisingly, perhaps, the progressors tended to have a UIP pattern and more severe ILD pre-treatment, with a predicted median DLCO of 42%. This cohort study demonstrated long-term efficacy with RTX treatment in terms of stabilising RA-ILD for many .A longitudinal multi-centre study from the Spanish NEREA registry published in 2020 also added to the RTX evidence base. This defined a very tight pulmonary “functional impairment” (FI) as a decline in FVC of ≥5%. Of the 68 RA-ILD patients, 31 received RTX. In total, 50% reached FI within 1.75 years of ILD diagnosis. However, multivariate analysis showed that RTX reduced the risk of FI by half compared with non-exposure . In terms of the RTX non-exposed group, the authors note that this group were treated with azathioprine (AZA), ABA and mycophenolic acid (MMF) in descending order of frequency. The take home message here is that RTX-exposed patients have a much higher probability of stabilising or improving compared with other forms of treatment .p = 0.02). RTX improved the FVC from baseline by 7.5% . The authors report that while their meta-analysis highlights a significant improvement in the FVC after one year of RTX treatment, the trend towards an improvement in DLCO is not statistically significant. They conclude that there remains an urgent need for further studies, including RCTs comparing the effectiveness of RTX against other immunosuppressants [A recent systematic review and meta-analysis examining the evidence for RTX use in RA-ILD identified 15 suitable studies in total: 11 retrospective and 4 prospective studies. Pooling the patients provided a total of 314 RTX-treated RA-ILD cases, with a heterogenous spectrum of disease representative of real-world clinical practice. The overall pooled proportion of patients with stabilisation or improvement in ILD was 0.88  [Early real-world experience with ABA showed promising results in RA-ILD patients. ABA has been available in Japan since 2010 and Nakashita et al. performed a retrospective single-centre cohort study comprising 16 ABA-treated RA-ILD patients and 46 TNFi-treated RA-ILD patients. They found that none of the ABA-treated patients showed HRCT evidence of progression one year after starting ABA treatment. This is in contrast to the TNFi group, in which 30.4% (14/46) of patients showed evidence of ILD progression on HRCT (< 0.013) . While t< 0.013) .The Italian experience with ABA mirrors the early Japanese cohort studies. Cassone et al. published their retrospective multi-centre cohort study involving 44 RA-ILD patients treated with ABA in 2020. Serial PFTs found that the FVC and DLCO remained stable or improved in 86% and 91% of patients over a median follow-up time of 26.5 months. The HRCT appearance also remained stable or improved in 81% of patients. Additionally, they noted that the previous or concurrent use of MTX had no effect on the outcome .Further evidence from Spain also shows a strong stabilising effect with ABA. Fernandez-Diaz et al. published a large national multi-centre observational study involving 263 RA-ILD patients treated with ABA. At baseline, this cohort had a median duration of ILD of 1 year, a predicted mean FVC of 85.9% and a predicted DLCO of 65.7%. After a median follow-up period of 1 year, the FVC and DLCO were stable in 87.7% and 90.6% of the cohort. The HRCT findings were stable in 76.6% of the cohort . FurtherCumulatively, the body of evidence for ABA’s role in RA-ILD treatment is amongst the strongest compared with other classes of immunosuppressant and should probably be considered alongside RTX as the first-line treatment.TCZ is an interleukin-6 (IL-6) inhibitor that was first approved for RA in 2009 in Europe by the European Medicines Agency (EMA) and in 2010 in the United States by the Food and Drug Administration (FDA). The data supporting the use of TCZ in RA-ILD are somewhat mixed. Curtis et al. performed a large retrospective cohort study in RA to investigate the incidence of ILD in patients receiving TNFi, IL-6 inhibitors or other bDMARDs acting on T-cells or B-cells. They found no significant difference in the incidence of ILD or hospital admissions for patients with known RA-ILD in 59 TCZ-treated patients compared with 232 TNFi-treated patients . InteresIf Nakashita’s study was interpreted as a win for TCZ in comparison to the TNFi class, then the picture was clouded by a post-marketing surveillance study from Japan of 7901 TCZ-treated RA patients, as the incidence of ILD was found to be 10 cases per 1000 patients per year. This is approximately twice as common as their estimated incidence of the disease . Is it tWhile TCZ may confer a protective effect against ILD in RA patients, there is insufficient evidence to use it ahead of either ABA or RTX. Prospective comparative studies are still needed to determine its relative efficacy to both ABA and RTX.Janus Kinase Inhibitors (JAKi) are oral novel small molecules and the newest class of RA therapeutics. To date, the paradigm for RA-ILD treatment has been tight disease control with the up-titration of MTX and the addition of bDMARDs such as ABA or RTX. More recently, small real-world cohort studies have suggested that the magnitude of the proposed beneficial effect of JAKi on RA-ILD is comparable to ABA in terms of HRCT improvement, stabilisation, and progression . AdditioJAKi has also demonstrated a greater reduction in KL-6 levels than other bDMARDs. Given that elevated KL-6 levels tend to correlate with the active fibrotic process, it is hoped therefore that this will result in a strong stabilising effect on ILD with JAKi treatment .p = 0.0090) compared with patients treated with ADA [The best supportive evidence for the potential beneficial role of JAKi in RA-ILD comes from a large retrospective cohort study involving over 28,000 RA patients in the United States. Patients treated with tofacitinib (TOFA) had the lowest incidence of ILD of all bDMARDs. After adjusting for covariates, TOFA still demonstrated a 69% reduced risk of ILD  in an anti-metabolite that inhibits T and B lymphocyte proliferation. It has been used since the early 1990s as a potent immunosuppressant in transplant medicine. It has been widely used in the treatment of connective-tissue-disease-related interstitial lung disease (CTD-ILD) for years, and particularly in diffuse systemic sclerosis lung (dSSc) involvement. The landmark Scleroderma Lung Study II (SLSII) showed that the treatment of SSc-ILD with MMF for 2 years or cyclophosphamide (CYC) for 1 year both resulted in significant improvements in pre-specified measures of lung function, dyspnoea, lung imaging, and skin disease over the 2-year course of the study. While MMF was better tolerated and associated with less toxicity, it was not more efficacious than CYC at the 2-year mark .While it is tempting to assume that MMF will have a beneficial effect on RA-ILD given its efficacy in other rheumatic conditions, it may not be ideal to promote the drug as a first-line therapy in RA-ILD given that there are various bDMARDs with a more robust evidence base in this domain. To the best of our knowledge, there are only four publications in the literature and a small number of MMF-treated RA-ILD patients that suggest its beneficial role.Saketkoo et al. described a small case series of just three patients with RA-ILD treated with MMF. The improvement in PFTs and HRCT stabilisation suggested a possible beneficial role . More inA retrospective UK study from 2016 reports a better survival rate for RA-ILD treated with MMF rather than with azathioprine (AZA). Notably, they found that the relative risk of death was increased with glucocorticoid (GC) treatment, unchanged in those treated with AZA, and reduced in those given MMF . Lastly,The take-home message that can be derived from the limited evidence base is that perhaps MMF has some stabilising effect on RA-ILD, but it is difficult to quantify the magnitude of the effect or compare it to other forms of immunosuppression used in RA on the basis of these findings. While we have various bDMARDs with a more robust evidence base, such as ABA and RTX, we should refrain from opting for MMF as a first-line treatment in RA-ILD. Prospective trials comparing ABA, RTX and MMF are needed to clarify this issue.p = 0.041). The efficacy of nintedanib versus the placebo was numerically greater in RA-ILD patients with a UIP pattern on HRCT compared with other fibrotic patterns, but this trend did not reach statistical significance. Furthermore, the rate of decline in FVC was slower in RA-ILD patients treated with nintedanib rather than the placebo regardless of whether or not they were treated with background DMARDs or steroids at baseline. This suggests some synergistic treatment effect in combining DMARDs and nintedanib [The role of anti-fibrotics in the treatment of RA-ILD still requires further clarity. The current evidence base suggests that anti-fibrotics have a supportive synergistic role as an adjunct to safely maximising MTX and a suitable bDMARD such as ABA, RTX or a JAKi. The utility of nintedanib has already been demonstrated in idiopathic pulmonary fibrosis (IPF) in two replicate 52-week RCTs, INPULSIS-1 and INPULSIS-2 . The INBntedanib ,47. NoneIn terms of other anti-fibrotic agents, the TRAIL1 study was a phase 2 double-blind RCT that aimed to assess the efficacy and safety of pirfenidone in RA-ILD patients. Unfortunately, TRAIL1 was disrupted by the COVID-19 pandemic and ultimately only 123 patients were recruited, despite requiring 270 for an adequately powered study. The primary endpoint was the composite of a greater than 10% predicted decline in FVC % or death during the 52-week study period. This endpoint was met by 11% of the pirfenidone group versus 15% of the placebo group, suggesting a favourable protective effect against RA-ILD deterioration. However, given the difficulty with recruitment, the trial was underpowered to detect a statistically significant distance between groups in the composite endpoint ,49. NeveUltimately, the body of evidence suggests that nintedanib likely has a synergistic effect when combined with DMARDs. The potential protective effect of pirfenidone in RA-ILD is a little less certain, although the trend is towards a beneficial effect. In practice, we would recommend the trial of an anti-fibrotic in those with progressive fibrotic lung disease, opting for nintedanib over pirfenidone, with commencement ideally after first monitoring the response to MTX and bDMARD . If nintedanib is poorly tolerated from a gastrointestinal standpoint despite the use of loperamide, pirfenidone may be an alternative.Unsurprisingly, careful consideration must be given to the choice of immunosuppression on a risk–benefit case-by-case basis. Pre-existing lung disease and a poor pulmonary reserve prior to treatment, along with advanced age, make this a frail cohort of RA patients particularly susceptible to iatrogenic or immunosuppression-related infections. While much of the sequalae of long-term glucocorticoids can now be avoided with all the therapeutic classes available today, there remains an increased risk of bacterial pneumonias and class-specific risks. RTX may be the most suitable agent in those with a history of recent malignancy, but it has a particularly potent deleterious effect on vaccination response; in addition, given the long-lasting effect of a single infusion, one must be cautious of its use in frail patients prone to recurrent infections. In this sense, ABA may be a better option, but there is still a rare clinical scenario in which a prior history of melanoma would make it a poor choice. Lastly, PJP is probably the greatest concern in this cohort of frail patients. It can be difficult to diagnose given that the clinical picture can be confused with ILD exacerbation or a bacterial or viral LRTI superimposed on background pulmonary disease. PJP is a serious and often underappreciated opportunistic infection in RA patients. While the incidence of PJP is lower in non-HIV patients, the associated mortality is considerably higher in RA-ILD patients on bDMARDs, at approximately 30% compared to 10–20% in HIV-positive patients ,53,54. PInterestingly, the rate of the asymptomatic carriage of P. jirovecii in the RA population ranges from 10.9% to 28.5% ,61,62. AFirst and foremost, the groups that benefit from PJP prophylaxis in general are those that are immunocompromised and are at a higher risk of PJP infection. Typically, PJP prophylaxis is recommended in HIV-infected patients with low CD4 cell counts, transplant recipients and those with haematologic malignancies. While the incidence rate of PJP is certainly lower in the RA-ILD population than in the aforementioned at-risk groups, the mortality rate in the RA-ILD cohort is alarmingly high. Katsuyama et al. showed that RA patients with two or three risk factors for PJP, such as steroid use, pre-existing lung disease and older age >65), benefited from primary prophylaxis . If init, benefitThere remains little by way of international consensus guidelines for the treatment of RA-ILD. The eagerly awaited American College of Rheumatology (ACR) guideline has yet to be formally published, but it has already raised eyebrows with its promotion of mycophenolic acid (MMF) as a first-line treatment and its heavy focus on nintedanib in the treatment algorithm. This guideline for the treatment of ILD in people with systemic autoimmune rheumatic disease (SARD) conditionally recommends MMF as the first-line therapy in RA-ILD, as well as the first-line treatment in all systemic-autoimmune-rheumatic-disease-associated ILD (SARD-ILD) . The finCurrently, we believe that the Spanish Society of Rheumatology (Sociedad Espanola de Reumatologia or SER) has created and promoted the most suitable guidelines for RA-ILD based on the current available evidence base for each drug. The SER promotes individualised assessment for the use of MTX given the risk of drug-induced pneumonitis. They also advise the continuation of MTX in patients who have already been receiving the drug for more than one year at the time of RA-ILD diagnosis, as there is no evidence to justify discontinuation. ABA and RTX are the recommended bDMARDs of choice if required, and the SER does not promote one over the other. If there is an inadequate response to ABA or RTX, the SER recommends a switch to either TCZ or a JAKi; the SER does not prefer one over the other. If GCs are considered necessary in inflammatory non-UIP radiological patterns , then their use is recommended at the lowest dose and shortest duration possible . Our proTo the best of our knowledge, the BSR is currently in the process of developing its guidelines for the treatment of RA-ILD. It will be interesting to see whether the BSR’s new guidelines fall more in line with the SER or the ACR. We highlight and compare some of the key recommendations from the ACR, SER and BTS in While autoimmune parenchymal lung disease remains a significant cause of morbidity and mortality in RA patients, it is now clear that MTX has a protective rather than detrimental effect on ILD development. The current evidence base supports the avoidance or cessation of TNFi in those with newly diagnosed ILD, and the commencement of ABA or RTX preferentially. There remains a role for high-dose GCs in the acute setting and particularly in the presence of either the NSIP or OP pattern on HRCT. The short-term use of PJP prophylaxis is reasonable while tapering high-dose GCs. The emerging evidence suggests that the addition of an anti-fibrotic, preferentially nintedanib, has a synergistic protective effect. Ideally, combination MTX and bDMARD treatment should be optimised and monitored with follow-up PFTs  before the addition of an anti-fibrotic. There remains a great need for further RCTs and prospective studies to help better determine the optimum treatment strategy in RA-ILD with the currently available drugs on the market.MTX has a protective effect in RA-ILD patients and should be continued.TNFi use should be avoided in RA-ILD patients.ABA and RTX have the strongest evidence base and either should be added to MTX as a first-line bDMARD.TCZ or JAKi may be considered as a second-line bDMARD in RA-ILD.MMF may be considered in combination with RTX in refractory disease.There remains a short-term role for high-dose GCs in NSIP or OP, but not UIP, patterns of RA-ILD.Long-term GCs should be avoided.PJP prophylaxis may be prescribed in the short term for a limited duration if on tapering high-dose GCs.Nintedanib may be added after the optimisation of combination MTX + bDMARD (ABA or RTX).Pirfenidone may be trialled if nintedanib + loperamide is not tolerated from a GI perspective.