DDX) can act either as promoters or suppressors in various cancer types. Nevertheless, the function of DDX49 in prostate cancer (PCa) is unknown. This study reveals the prognostic and predictive value of DDX49 in PCa.There is increasing evidence that DEAD-box helicases (DDX49 between PCa and normal tissues based on TCGA and GEO databases. Univariate and multivariate regression analyses were conducted to reveal the risk factors for PCa recurrence. A K–M curve was employed to assess the relationship between DDX49 and recurrence-free survival. In vitro, DDX49 expression was evaluated in PCa and normal prostate cell lines. Furthermore, we constructed a shDDX49 lentivirus to knock down the expression of DDX49. Celigo® Image Cytometer and MTT assay were performed to analyse cell proliferation in PC-3 cells. Cell cycle distribution was detected with flow cytometry analysis. Apoptosis affected by the lack of DDX49 was metred with the PathScan® Stress and Apoptosis Signalling Antibody Array Kit.First, we evaluated the expression of DDX49 in PCa tissues in comparison with normal tissues and that increased DDX49 indicates a poor prognosis among PCa patients. Meanwhile, DDX49 knockdown suppressed the proliferation and migration of PC-3 cells, causing cell cycle arrest in the G1 phase. Stress and apoptosis pathway analysis revealed that the phosphorylation of HSP27, p53, and SAPK/JNK was reduced in the DDX49 knockdown group compared with the control group.This study shows a high increase in DDX49 predicts a poor prognosis among PCa patients. Downregulation of DDX49 can suppress cell proliferation, block the cell cycle, and facilitate cell apoptosis. Therefore, knockdown of DDX49 is a promising novel therapy for treating patients with PCa.In summary, these results suggest that high expression of The online version contains supplementary material available at 10.1186/s12894-023-01251-4. Prostate cancer (PCa) is among the dominant causes of cancer deaths in men worldwide . AndrogeDDX1 was involved in the initial progression of testicular tumours through the regulation of cyclin-D2, CD9 and NANOG [DDX1 promotes tumorigenesis and that DDX1 is associated with the high expression of MYCN in advanced neuroblastoma, which indicates a poor prognosis [DDX9 has been reported among many cancers, and elevated DDX9 may promote Wilms tumour metastasis, contribute to the progression of colorectal cancer through the activation of the NF-κB signalling pathway, and be related to the poor clinical outcome of lung cancer [DEAD (Asp-Glu-Ala-Asp)-box family genes exert a vital function in the metabolism and functional activation of RNAs . Previound NANOG . Furtherrognosis . Overexpg cancer –14.DDX in PCa. Clark et al. found that DDX5 is overexpressed in PCa compared to normal prostate tissue, and DDX5 expression is positively correlated with the stage of PCa. More importantly, they demonstrated a possible functional role for DDX5 as an androgen receptor (AR) transcriptional coactivator and mediator of posttranscriptional regulation of AR mRNA [DDX3 also regulated AR posttranscriptionally by sequestering mRNA, and inhibiting DDX3 was sufficient to reduce AR protein expression and signalling, resensitizing PCa cells to AR signalling inhibitors [DDX52 knockdown inhibits PCa cell growth by regulating c-Myc signal transduction, and they found that DDX52 has a significant decreasing effect on c-Myc, which promotes the progression of PCa and that c-Myc’s oncogenic potential in PCa cells may rely on DDX52. However, the DDX family comprises at least 35 genes, most of which have not been evaluated for function in PCa, especially in castration-resistant PCa. It is important to understand how these genetic changes contribute to the progression of PCa, as they will contribute to the study of the mechanism and treatment of PCa.Furthermore, several studies have also investigated the roles of  AR mRNA . In anothibitors . Yu et ahibitors  reportedDDX49 and PCa prognosis in the Cancer Genome Atlas (TCGA) as well as the Gene Expression Omnibus (GEO) database. Then, lentivirus-delivered short hairpin RNA (shRNA) was applied to knock down DDX49 in PCa cells, and changes in cell proliferation, the cell cycle and cell apoptosis were evaluated.Here, we reveal the association of DDX49 expression and PCa, six cohorts were enrolled from TCGA  and GEO databases (https://www.ncbi.nlm.nih.gov/geo/), including TCGA-PRAD, GSE29079, GSE25183, GSE46602, GSE38241, and GSE69223. From these, the association between different DDX49 expression levels and recurrence-free survival (RFS) in PCa patients was assessed. In addition, the correlation of DDX49 expression with apoptosis-related genes was assessed based on TCGA cohorts via a visualization tool, GEPIA (http://gepia.cancer-pku.cn/) [DDX49 expression from the STRING database, which predicted protein–protein interactions [DDX49 were analysed for cellular processes and pathways through Gene Ontology (GO) term enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.To explore the correlation between ractions . SubsequDDX49 and low-DDX49 groups in accordance with the mean DDX49 expression. The R package “limma” was employed for screening differentially expressed genes (DEGs) between groups with the absolute value of fold-change > 0.4 and p adjusted value < 0.01 as cut-offs [Patients in the TCGA-PRAD cohort were classified into high-cut-offs . GO and cut-offs , 22.®, Shanghai, China), respectively. We cultivated cells at 37 °C and 5% CO2 under a humidified environment.We purchased human cell lines, including RWPE-1, PC-3, LNCaP, and C4-2, from the Shanghai Cell Bank . Keratinocyte serum-free medium (K-SFM) supplemented with human recombinant epidermal growth factor (EGF) and bovine pituitary extract (BPE) was adopted for culturing RWPE-1 cells. PC-3, LNCaP, and C4-2 cells were cultured with RPMI 1640 media, 10% foetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin–streptomycin liquid  was adopted for isolating total RNA from RWPE-1, PC-3, LNCaP, and C4-2 cells. In addition, the primers used to test the expression of DDX49 ORF was targeted to express shRNA using pGCL-GFP-lentivirus (GenBank no. NM_003410) (shDDX49 lentivirus). A nontargeting sequence was used for the lentivirus-negative control (shCtrl) . Experiment template: 5′-GCCTGAGAATGATCATGGA-3′.The We chose PC-3 as the candidate for treatment with shDDX49 lentivirus. According to the MOI of lentivirus, 5 µl of shDDX49 or shCtrl lentivirus was added to each well of PC-3 cells cultured in 6-well plates. After the first 72 h of infection, we observed green fluorescence.® Image Cytometer to automatically identify the intensity and distribution of fluorescence in the PC-3 cells after treatment with shCtrl or shDDX49 lentivirus. After treatment with lentivirus for ten days, PC-3 cells in different groups were inoculated on 96-well plates at 2000 cells per well, and in the following five days, Celigo® Image Cytometers were adopted to record the cell status each day.We adopted the CeligoDDX49. PC-3 cells in different treatment groups were inoculated on six 96-well plates at 2000 cells per well, and we collected and detected the activity of cells on the 1st, 2nd, 3rd, 4th, and 5th days. First, the cells were washed three times in cold PBS, and a 3--2,5-diphenyltetrazolium bromide (MTT) solution was added to every well to reach a final concentration of 0.5 mg/ml. Then, the cells were cultured in MTT solution at 37 °C for 4 h, and the solution was discarded. Finally, formazan salt was solubilized in 100 ml of dimethyl sulfoxide (DMSO) for 10 min. In addition, the OD was measured at 490 nm per well.The MTT assay is another way to evaluate cell growth inhibition caused by the lack of We employed the flow cytometry system to assess the different proportions of cell cycle stages among the shCtrl and shDDX49 groups. After treatment with different lentiviruses, we collected the cells at 1, 2, 3, 4, or 5 days. Cells were rinsed twice in cold PBS, fixed with 0.5 ml of 70% ethanol, and cultured for 1 h at 4 °C. Then, 50 µg/ml propidium iodide (Sigma‒Aldrich) was used to stain these cells. The cells were filtered by a 300-mesh filter, and then we recorded the cell cycle phase of each cell through the stained nuclei using a BD FACSCalibur flow cytometer .DDX49 affects the apoptosis pathway, the PathScan® Stress and Apoptosis Signalling Antibody Array Kit was used. This kit tested the phosphorylation statuses of ERK1/2, Akt, Bad, HSP27, Smad2, p53, p38 MAPK, SAPK/JNK, Chk1, Chk2, IkBa, eIF2a, and TAK1, the cleaved levels of PARP, cleaved caspase-3, and cleaved caspase-7, and the total expression levels of IkBa, survivin, and α-tubulin. First, after 48 h of transfection with shDDX49 or shCtrl lentivirus, we collected and washed the PC-3 cells twice with cold PBS. The cell protein concentrations were regulated to 0.2–1.0 mg/ml. After treatment with blocking buffer for 15 min, all 50–75-µl cell protein samples were introduced into every tube and cultured at 4 °C overnight. On the second day, the cell protein solution was erased, and the plate was rinsed three times for 5 min each and then treated with 75 µl of 1 × antibody while gently shaking for 1 h at room temperature. Then, 75 µl of 1 × HRP-streptavidin was added for 30 min. Finally, the plate was washed three times and then exposed and visualized on the CLINX ChemiScope 5300 machine.To evaluate whether the lack of DDX49 expression with clinicopathological characteristics, Fisher’s exact test and Pearson’s chi-squared test were conducted, while Student’s t-test was adopted to assess the differences between groups. Recurrence-free survival (RFS) was analysed with the Kaplan–Meier method, and differences were assessed with the log-rank test. Multivariate recurrence-associated analysis was carried out with logistic regression analysis. The relative risks of PCa recurrence were denoted as adjusted hazard ratios and corresponding 95% confidence intervals (95% CIs). The statistical data are displayed as the mean ± SD. For all analyses, p < 0.05 indicated statistical significance. In addition, SPSS version 22.0  was involved in statistical analyses.To explore the association of DDX49 between PCa tumour tissues and normal tissues were compared based on the TCGA-PRAD cohort, which contained the transcription profiles of 497 tumour tissues and 52 normal tissues. DDX49 was significantly increased in PCa tumour tissues (p < 0.001) , GSE25183 (p = 0.0031), GSE46602 (p = 0.0030), GSE 38,241 (p < 0.0001), and GSE69223 (p = 0.0165), and found increased DDX49 expression levels in PCa tissues compared with normal tissues . Similar results were observed in patients with PCa categorized as T3/T4 (p = 0.003) or N1 (p = 0.126) . Consistently, patients in the recurrence group showed an increased risk of advanced PCa, judged by pathology T (p < 0.001), N (p = 0.002), and M (p < 0.001) stages. Meanwhile, elevated expression of DDX49 was found in patients with recurrent PCa compared with patients who were free of recurrence , indicating that DDX49 may be involved in the biochemical recurrence of PCa. PCa tissues were further classified into high-expression and low-expression groups on the basis of the median expression level of DDX49, and K–M curves were used to compare the different RFS between groups. Based on the TCGA-PRAD cohorts, we found that RFS was shorter among patients with high DDX49 expression than among patients with low DDX49 expression ; this was also confirmed in the MSKCC cohorts . Additionally, multivariate logistic regression analysis was conducted to enrol all the clinical features and DDX49 expression, and the results indicated that Gleason score , pathology T stage , and DDX49  were independent risk factors related to PCa recurrence among patients  , whereas the distributions of G2/M and S phase cells were dramatically decreased (p < 0.01).This study further evaluated the impacts of DDX49. We found that the phosphorylation levels of HSP27, p53, and SAPK/JNK were decreased in the shDDX49 group compared with the shCtrl group. Likewise, cleaved caspase-7 was also decreased in the shDDX49 group , p53 , SAPK , and CASP7  (Fig. By adopting the PathScan® Stress and Apoptosis Signalling Antibody Array Kit, we detected changes in stress and apoptosis signalling markers caused by suppression of oup Fig. A. AdditiERG, ETV1/4, FLI1, SPOP, FOXA1 and IDH1; however, prior studies are still insufficient to cover all patients with PCa, and nearly 26% of available PCa tissue samples require further study to investigate their gene signatures [DDX49 as a potential prognostic gene for PCa.In recent decades, PCa has been considered the dominant cause of cancer-specific death among males . AlthougDDX gene belongs to the helicase 2 superfamily and is involved in almost every aspect of RNA metabolism [The tabolism , 28. Thetabolism . A growitabolism , 31. TheDDX49, a member of the DEAD-box RNA helicase protein family, has been shown to maintain stable levels of preribosomal 47S RNA and regulate global translation [DDX49 may be hijacked in cancer to promote uncontrolled cell proliferation, consistent with numerous studies. Lian et al. [DDX49 in lung adenocarcinoma and cell lines in comparison with paracancerous tissues and normal cells, and DDX49 knockdown suppressed the viability and invasion of lung cancer cells. DDX49 expression among breast cancer patients was also markedly increased. In addition, DDX49 may promote the growth of breast cancer stem cells by regulating the expression of Oct3/4, SOX-2 and other proteins, thus promoting the development of the disease [DDX49 is overexpressed in hepatocellular carcinoma and leads to poor prognosis [DDX49 and the prognosis of PCa has not been explored until now.nslation . This prn et al.  discover disease . In addirognosis . HoweverDDX49 and the recurrence of PCa and that DDX49 is predictive of a poor patient prognosis. Additionally, the proportion of PC-3 cells arrested in G1 phase was significantly enhanced after DDX49 knockdown. As cell cycle arrest reflects the alteration of apoptosis [DDX49 expression on the protein pathways regulating apoptosis. DDX49 knockdown decreased the phosphorylation abundance of p53, an important protein that is involved in regulating apoptosis in PCa [DDX49 on PCa cells. Furthermore, the phosphorylated form of HSP-27 was decreased after DDX49 knockdown. Interestingly, previous reports have confirmed that Hsp-27 can promote the occurrence of CRPC by protecting eukaryotic translation initiation factor 4E from degradation [DDX49 may not only play a cancer-promoting role in prostate cells by affecting p-HSP27 but also exert significant effects on CRPC development via p-HSP27. In this study, we also showed a decreased level of SAPK/JNK phosphorylation, indicating that SAPK/JNK may participate in the cancer-promoting function of DDX49 in PCa. However, the exact mechanism by which DDX49 affects the phosphorylation abundance of these proteins is unclear. Recent studies have shown that DDX3 can bind to casein kinase 1 as a regulatory subunit to directly stimulate its kinase activity and promote protein phosphorylation [DDX49 may promote the phosphorylation of these proteins in a similar way. Further work is needed to elucidate how DDX49 affects these possible pathways to modulate the cell cycle, apoptosis, and cell proliferation.This research first confirmed the relationship between high expression of poptosis , we nexts in PCa . We specradation . This rerylation . We specDDX49 expression with PCa recurrence, which is predictive of a poor patient prognosis. In vitro results also demonstrated that the downregulation of DDX49 expression suppressed cell proliferation, blocked the cell cycle, and facilitated cell apoptosis. Therefore, knockdown of DDX49 may become a potential new strategy to treat PCa patients.In summary, this study demonstrates the association of high Additional file 1: Table S1. The selection of DDX49 associated gene from the STRING.Additional file 2: Table S2. The GO and KEGG enrichment results of DDX49 related genes.


Editorial on the Research TopicPerioperative organ dysfunction and protection in neonates and preterm infantsGe et al.) report the rate of HDP among mothers of very preterm infants (VPI) in the Chinese Neonatal Network. This article perhaps shines new light on the effect of HPD on neonatal outcomes.Globally, there are approximately 2.3 million neonates who die in the first 28 days of their life from various causes. Prematurity secondary to maternal hypertensive disorder of pregnancy (HDP) remains a leading cause of very preterm birth. In this Research Topic in Frontiers in Pediatrics,  report a prospective cohort study on the incidence of death and its predictors among neonates admitted with sepsis to a referral hospital in northwest Ethiopia. They find low birth weight, prematurity, duration of labor longer than 24 h, not breastfeeding, having respiratory distress syndrome (RDS), and oxygen saturation lower than 90% as being the significant predictors of death among neonates admitted with sepsis. They diagnosed sepsis by questionaries rather than blood culture. Their findings are more relevant to low-income countries.Not every country has the resources of China or Western countries. In this Research Topic in Frontiers in Pediatrics,  reports an 82.83% incidence of hypothermia intraoperatively despite the application of forced warm air mattresses and warmed intravenous fluid. It illustrates that there is still a lot of room for improvement. They further describe whether a cuffed endotracheal tube or low fresh gas flow technique were utilized and whether the setting of Operation Room temperature might have helped to pinpoint the causes for this high intra-operative hypothermia rate.For some neonates, surgical intervention is required in the newborn period. One of the leading causes of perioperative complications is intra-operative hypothermia. Not only can hypothermia lead to catecholamine surges, increased metabolism, and vasoconstriction, but also leads to prolonged post-operative extubation times and surgical site infection. Despite the wide recognition of these detrimental effects, intra-operative hypothermia, defined as a temperature lower than 36°C, is still alarmingly high. In this Research Topic in Frontiers in Pediatrics, the retrospective observation study by Lai and colleagues  present a review of this topic. Another common peri-operative complication, in particular for those neonates receiving intra-abdominal procedures, is acute kidney injury (AKI). In this Research Topic in Frontiers in Pediatrics, Duong and colleagues (From better-controlling hypertension during pregnancy and the early detection of sepsis among neonates, to maintaining normothermia intraoperatively and the prevention or mitigation of AKI, there seem to be a lot of opportunities for clinicians tasked with the care of neonates.


A new Irish bovine tuberculosis (bTB) eradication strategy was launched in 2021. The strategy was formulated following extensive discussions with stakeholders, formal reviews of several aspects of the existing bTB policy and relevant inputs from the latest scientific research projects. A stakeholder discussion body, the TB Forum, had been established in 2018 and this continues under the new strategy, supported by three working groups . The strategy sets out actions to address cattle-to-cattle and badger-to-cattle bTB transmission, along with actions to improve farm biosecurity and empower farmers to make their own choices to reduce bTB risk.2 covered by the vaccination programme and 6,586 badgers captured in vaccination areas in 2021. Vaccination efforts have been complemented by intensive communications campaigns, including a web enabled software application (“app”) enabling farmers to report the location of badger setts.Large scale vaccination of badgers has been rolled out under the new strategy, with over 20,000 kmCattle which test inconclusive to the tuberculin skin test have been re-tested using a gamma interferon blood test since April 2021, enabling truly infected cattle to be identified more effectively due to the higher sensitivity of this test. An enhanced oversight process has been put in place for herds experiencing extended or repeat bTB breakdowns. Whole genome sequencing is being used to investigate links between breakdowns, with the results supporting operational decision making in case management.Communications, including biosecurity advice, are co-designed with stakeholders, in order to improve their effectiveness. A programme involving veterinary practitioners providing tailored biosecurity bTB advice to their clients was established in 2021 and was rolled out nationally during 2022.A core element of the new strategy is the continual improvement of policies in response to changing bTB risks, informed by scientific research and then implemented with stakeholder consultation. Mycobacterium bovis, continues to pose a significant animal health challenge in Ireland. The objective of this paper is to describe advances within the Irish bTB eradication programme in recent years and to set out the structural relationships between scientific research, stakeholder attitudes and policy development. The paper also seeks to provide a wider historical perspective on the Irish bTB programme, putting the recent changes and strategic developments in context.Bovine tuberculosis (bTB), caused by Current bTB levels in Ireland impose significant direct and indirect costs on the public and private sectors and are a threat to Irish export market access. In 2021, national herd incidence was 4.33%, with 20,931 cattle identified as TB test reactors. Direct costs in 2021 were estimated at €105 m, with €67 m paid by the State, an estimated €35 m paid by farmers and €3 m by the EU; these figures do not include indirect costs or production losses. The costs of bTB in Ireland are not confined to financial issues; restrictions applied to infected herds can cause significant emotional distress to herdowners [An eradication programme for bTB is required under EU legislation, with the rules for countries or zones where disease is present set out in Regulation (EU) 2016/429  and Commission Delegated Regulation 689/2020 (which sets out the specific requirements for bTB). The Irish bTB programme applies these rules, as well as additional national requirements relevant to the epidemiological context in Ireland.The Irish bTB eradication commenced in 1954, with tuberculin skin testing used in all herds to identify infected cattle, which were then removed and slaughtered. At that point, it was estimated that 80% of herds and 17% of cattle (22% of cows) were infected . By 1992A major bTB research programme was established in 1991, principally involving researchers employed within the Department of Agriculture, Food and the Marine and University College Dublin School of Veterinary Medicine, with the aim of providing an evidence base for new policies and strategic decisions . ResearcA significant and ongoing research programme on the use of Bacille Calmette-Guerin (BCG) vaccination in badgers to reduce the risk of badger-mediated bTB transmission to cattle was a key element of this overall research programme ; this reThe challenge of improving diagnostics for bTB was also a focus of much research. This included work on the tuberculin test –28, the By 2016, bTB levels in Ireland had reached an all-time low, with herd incidence at 3.27%. However, the removal of milk quotas in 2015 prompted the expansion of the dairy sector in Ireland. Dairy herds, particularly the larger ones, have many risk factors for bTB, and thus have a higher disease incidence compared to other cattle enterprises. The expansion of the dairy sector was therefore associated with an increase in bTB. Consequentially, bTB levels rose each year until they peaked at 4.38% in 2020. The confluence of improved scientific understanding, the availability of new tools, increasing disease levels, and concerns about the ongoing costs and burden of bTB prompted an initiative to renew the Irish bTB strategy in 2018.www.bovinetb.ie [The TB Forum was established in 2018 to discuss bTB and make recommendations to the Minister for Agriculture, Food and the Marine on additional measures which could be implemented to reduce bTB levels and further the drive towards eradication. The Forum was composed of a range of stakeholders and submitted a report on its recommendations in 2019. This report, the policy analysis document which informed its development, and the minutes of the TB Forum meetings, can be accessed at inetb.ie . Two repinetb.ie . Followiinetb.ie .The framework for considering the range of bTB issues was for policies to be reviewed and discussed at the TB Forum and also subjected to ongoing scientific research and policy analysis. This was often an iterative process, with the results of research informing an updated policy analysis, leading to proposals which, if implemented, would then be the subject of further scientific research. These steps were not necessarily sequential but could progress in parallel, overlapping temporally. The TB Forum provided a vehicle for the frequent exchange of policy information, scientific results, and stakeholder opinions. The policies which were introduced or updated as a result of this process, and the manner of their implementation, were then the subject of ongoing discussion at the TB Forum. In this way, a network approach to policy development was employed, enabling stakeholders to participate in the co-design of policies and the consideration of challenging choices between policy alternatives.M bovis has been the subject of a considerable body of scientific research in recent decades, particularly in the UK and Ireland. Many studies investigated the potential of orally-administered BCG to generate protective immunity in badgers [The idea of using Bacille Calmette Guerin (BCG) to vaccinate badgers and thus reduce badger-to-cattle transmission of  badgers –38. DespA major field trial was carried out in Co. Kilkenny on field vaccination of badgers , 40. A sResearch on vaccination was complemented by the badger ecological research mentioned above, enabling the design of a vaccination programme which could be implemented nationally in the field. The knowledge base was further progressed by studies carried out in Northern Ireland which examined similar topics in a similar ecological situation , 43.In 2018, routine vaccination of badgers using injectable BCG was established as policy within the Irish bTB eradication programme. Initially, vaccination was carried out in areas which had been part of the field research programmes, with incremental expansion to additional areas over time. The 2019 Programme for Government included a commitment to extend the badger vaccination programme nationwide and end badger culling as soon as possible, consistent with the best scientific and veterinary advice.2 in Ireland was designated a badger vaccination area; this is more than half of the total area on which the DAFM wildlife unit operates. In these areas, vaccination is the default, although culling may still be carried out where required for epidemiological reasons. In 2021, the DAFM wildlife unit captured 6,586 badgers in vaccination areas, of which 3,958 were then vaccinated (badgers captured which are found to have been previously vaccinated are not re-vaccinated), and captured 5,868 in culling areas  and Ingenasa ),) were used on badgers prior to vaccination; badgers which tested positive were culled and sent for post-mortem examination and culture. This work is ongoing and the results are expected to be published when complete. A second innovation was the development of a mobile phone app whereby farmers could report the location of badger setts to DAFM, to improve the population penetration of the vaccination programme, a key metric for effective badger vaccination . This apThe effectiveness of the badger vaccination programme and its impact on local transmission dynamics are the subject of an ongoing research project . The interim results of this work are regularly presented to stakeholders in the context of the TB Forum, providing for a degree of stakeholder confidence in the programme and demonstrating the continued commitment to evaluating and reviewing policies, including the badger vaccination programme.Cattle which test inconclusive to the SICCT test were required to be retested 42 days later, or slaughtered and subjected to laboratory examination or a balance of herd test in line with the requirements of Directive 64/432. If the repeat test disclosed them as inconclusive again or as positive, they were deemed reactors; while if negative, they were deemed to have passed the retest. Research had confirmed that in Ireland, such cattle which pass the retest continue to pose an elevated risk of being infected , 46. BasAt the TB Forum, stakeholders raised concerns about the ongoing risk posed by such retained inconclusives; while confined to the herds of disclosure for life, they could still initiate a repeat breakdown within those herds which may spread onwards. In response to these concerns, an updated policy on inconclusives was developed and implemented, informed by the earlier research. Policy analysis revealed that approximately 3,500 cattle annually tested inconclusive, with roughly 20% of these failing the retest.The new policy required cattle which tested inconclusive to the SICCT test to be subjected to an interferon gamma blood test within three weeks. If positive, the animal was deemed a reactor and removed. If negative, the animal was still required to undergo a further SICCT test. The interval for this repeat skin retest was increased from 42 to 60 days in order to counter the desensitising effect of tuberculin injection. If the animal passed this, it would require a further interferon gamma test six months later.Other related policy changes included the mandatory removal of any previously disclosed historic inconclusives  from a herd if one or more reactors was disclosed at a test; any cattle newly testing inconclusive to be deemed reactors if one or more standard reactors were disclosed at the herd test; and where four or more cattle tested inconclusive at a herd test with no standard reactors, all four were to be deemed reactors and removed and the herd restricted. In 2021, a letter was sent to the owners of all historic inconclusives, advising them to cull these cattle if they wished to reduce the bTB risk to their herd.In the first full year of operation of the new policy, 61% of inconclusive cattle which were interferon gamma tested were positive and thus removed as reactors, with the herd restricted. Within six months of the issuing of the advice letter on historic inconclusives by DAFM referred to above, 26% of historic inconclusive cattle had been slaughtered . The number of cattle reported as tested inconclusive to the skin test was 1,497 in the first full year since the new policy was introduced, a major reduction compared to the roughly 3,500 reported annually in years prior to the new policy.The subject of herds which experience extended and/or repeated bTB breakdowns has been the focus of considerable epidemiological research in Ireland –51. The Stakeholders are aware of the differences in seriousness between breakdowns based on number of reactors and length of restriction, and the subject of how better to manage such cases was discussed at the TB Forum. Informed by the research findings cited above, case management policy was updated to emphasise the progressive removal of all sources of bTB risk from extended and/or repeat breakdown herds. For example, if such a herd is not going clear, cattle whose test result is deemed “severe inconclusive” are removed, followed by cattle with a bovine bias in the SICCT, followed by cattle considered at higher risk due to their history of exposure. The policy discussions prompted a specific research project on the relationship between the bovine tuberculin response and future risk , the resM. bovis in reactor cattle [This topic is closely linked to the broader issue of effective breakdown management, where again new research has informed policy considerations. For example, research on faecal shedding of r cattle  underpinAll cattle slaughtered in Ireland are subjected to ante- and post-mortem inspections by a veterinarian, as required under EU food hygiene legislation. This includes checks for lesions which are suspected of being caused by bTB. Such lesions, termed “suspect factory lesions” in Ireland, are sent for laboratory testing and, if positive, the infected herd is restricted. Approximately one third of breakdowns in Ireland annually are first identified through surveillance at slaughter. Given its critical role in bTB surveillance in Ireland, this area has been the subject of a number of research studies , 55, witThe primary diagnostic test for detecting bTB in Ireland is the SICCT. The characteristics of this test make it the most suited option for a mass-screening programme, given its very high specificity . HoweverThe use of the interferon gamma test was introduced as policy for serious bTB breakdowns (defined as four or more reactors) in 2015, following several years of research in Irish field conditions , 32. TheThe tuberculin used in the programme has long been recognised as a key element requiring quality control and ongoing evaluation, with research showing the importance of using tuberculin of sufficient potency in the context of an overall quality control programme on bTB testing –28.Effective communication is crucial to the success of disease eradication campaigns, and the stakeholders at the TB Forum had signalled that improving communications to herdowners should be considered a priority. This was supported by research carried out on the attitudes of Irish farmers to the bTB programme .The subject of risk communication can be particularly challenging; in 2020, each cattle herd was assigned a TB herd history risk (TBHHR) status, which described if a herd was clear (C) or infected (INF), how many full years it had been clear and the number of bTB breakdowns in the preceding 10 years. For example, a TBHHR score of C4(2) means a herd has been clear for four full years and has had two bTB breakdowns in the past ten years. Figure The conflict between the interests of the buyer in avoiding introducing potentially bTB-infected cattle into their herd versus the interests of the seller of bTB-exposed cattle in maximising the price paid has been addressed by previous reviews of bTB in Ireland, including as far back as 1991  and a 19The introduction of a biosecurity advisory module on bTB which can be delivered by veterinary practitioners to their clients, funded by DAFM through Animal Health Ireland as part of their Targeted Advisory Service on Animal Health , has enaThese actions recognise the complex challenge of engaging with stakeholders on risk communication and advocating risk-reducing behaviour, which has been identified as an important area yet one in which much work remains to be done –63.www.bovinetb.ie) and partnering with other agricultural and veterinary advisory bodies to deliver training and information.Other communications efforts established in recent years to engage with farmers include the participation of DAFM bTB team members in many public meetings across Ireland, media engagement including podcasts, radio and television, creating a series of youtube videos and leaflets on specific topics of recurring interest  of deer submitted from the other 25 counties of Ireland were positive.In response to stakeholder concerns, the policy on deer and bTB was updated such that, if herdowners in an area were concerned that deer locally may be infected with bTB and playing a role in spreading disease to cattle, they could submit deer carcases to DAFM Regional Veterinary Laboratories, which would then test those carcases for bTB free of charge and report the results locally and to the TB Forum. This work was not, therefore, a random sample of deer; rather, only deer in areas where there were thought to be significant bTB levels in cattle were submitted for testing. Between 2016 and 2020, 35 of 272 (12.8%) of deer submitted from Co. Wicklow were positive for M bovis from badgers to cattle in the area consequent to the initiation of construction.Stakeholders at the TB Forum also conveyed the concerns of some of their members that activities which could disrupt wildlife, specifically the construction of roads and the clear-felling of forestry, could lead to an increase in local bTB levels in cattle. Epidemiological research was thus carried out into these questions, with the results shared with stakeholders. Researchers reported evidence of an increased bTB risk in cattle being consequent to, not just coincident with, road construction, and hypothesised that perturbation of badger populations may have provided the mechanism for this effect . AnotherIn relation to clear-felling of forestry within a wider ecological landscape context and bTB risk, evidence was found of an increased bTB risk following clear felling of forestry , but theThe Scientific Working Group of the TB Forum considered the issue of the sources of bTB infection for cattle, including the possible role of deer; this opinion was presented to stakeholders at the TB Forum and has been published online .M bovis cultures are being analysed using WGS in Ireland, principally isolates from cattle and badgers but also from other species such as deer, pigs and alpacas. The results are used to generate a wider national perspective on the distribution of M bovis strains in Ireland, and also to inform specific operational case management decision making and to address particular epidemiological queries at local level. The use of WGS as a tool to support epidemiological investigations and case managements illustrates how technical advances, when linked to the field management of cases, can provide more evidence for the case manager to take into account, with the objective of improving decision making. A detailed analysis of the results of this initiative will be reported separately. The scientific working group of the TB Forum has considered how WGS can best be used to support improved effectiveness in the Irish bTB programme, and their guidance will inform policy development in this area.The use of whole genome sequencing (WGS) has become widespread in bTB epidemiology in recent years , 70, 71,The introduction in March 2020 of societal restrictions to protect public health in relation to Covid19 in Ireland necessitated a rapid consideration of how the Irish bTB eradication programme could be adapted such that the public health rules were complied with while protecting animal health and enabling the continuation of cattle trade, considered a vital part of the Irish food supply system.Throughout the pandemic period, there was ongoing and frequent communication between the DAFM bTB team and stakeholders, particularly those representing farmers, veterinarians, and cattle marts. As the public health restrictions changed over the course of the pandemic, so too did the changes applied to the bTB programme, following discussions on each occasion with stakeholders. This illustrates the value of setting in place structures, such as the TB Forum, for ongoing engagement between policy makers and stakeholders.M bovis. A study was rapidly carried out on the risk factors related to the likelihood of a herd with a suspect lesion in a slaughtered animal subsequently having a bTB breakdown [At the start of the pandemic, DAFM’s high containment TB laboratory became a site for Covid19 testing. Although the consequent interruption to bTB testing turned out to be brief, concerns arose at the time that there could be a prolonged interruption to bTB laboratory culture capacity, due to the use of the laboratory for Covid19 testing and due to the risk of the highly experienced laboratory staff becoming unavailable if they became infected. The question arose of how to manage herds which had cattle with suspect factory lesions, if those lesions could not be cultured and tested for reakdown . Based oGuidance was provided to herdowners and veterinarians in relation to conducting the SICCT while adhering to the public health rules. Herd tests could be postponed for up to 28 days past the due date without penalty, to allow for circumstances where one party may have suspected or confirmed Covid19 infection, or where it proved challenging to obtain help from normal sources to assist with the test on the scheduled date. Small calves are usually held by one person while the veterinarian conducted the SICCT . To allow for social distancing guidelines to be adhered to, a temporary easement was allowed whereby calves aged 42–120 days could be excluded from a herd test if either the herdowner or the veterinarian were of the opinion that they could not be tested while adhering to the Covid19 rules. This easement was removed as Covid19 restrictions and social distancing guidance were changed. A review of the epidemiological considerations and risks related to bTB in calves was carried out to support policy development during this period; this work has been published and describes the topic in more detail .Machine learning approaches to predicting bTB risk have been described . A similThe scientific working group of the TB Forum has completed a consultation on future bTB research priorities with stakeholders from farming, veterinary and research backgrounds. Following the consultation, replies were systematically evaluated and priorities for future research were identified and presented to the TB Forum. These will inform the likely direction of bTB research in Ireland over the coming years.The wider international context will also continue to inform the development of Irish bTB policy. The bTB programme in Ireland sits within the legal framework of the EU regulations on the eradication of bTB, while the attitudes of trade partners constitute a major consideration for Irish policy makers. Technical and scientific developments in relation to bTB will also influence the Irish programme, with relevant innovations considered regarding the Irish epidemiological context.The Irish bTB eradication strategy will continue to change, adapting in response to new risks and new circumstances and seeking to make use of new tools as they become available. The paradigm of developing policy based on scientific research and through engagement and discussion with stakeholders provides a responsive and flexible model for ensuring that the programme can effectively tackle bTB in Ireland, driving towards an ultimate goal of eradication.


Oral diadochokinetic (DDK) tasks are common research and clinical tools used to test oromotor skills across different age groups. They include alternating motion rate (AMR) and sequential motion rate (SMR) tasks. AMR tasks involve repeating a single syllable, whereas SMR tasks involve repeating varying syllables. DDK performance is mostly discussed regarding the increasing rates of AMR and SMR tasks from childhood to adulthood, although less attention is given to the performance differences between SMR and AMR tasks across age groups. Here, AMR and SMR syllabic rates were contrasted in three populations: 7–9-year-old children, 14–16-year-old adolescents and 20–30-year-old adults. The results revealed similar syllabic rates for the two DDK tasks in children, whereas adolescents and adults achieved faster SMR rates. Acoustic analyses showed similarities in prosodic features between AMR and SMR sequences and in anticipatory coarticulation in the SMR sequences in all age groups. However, a lower degree of coarticulation was observed in children relative to adults. Adolescents, on the contrary, showed an adult-like pattern. These findings suggest that SMR tasks may be more sensitive to age-related changes in oromotor skills than AMR tasks and that greater gestural overlap across varying syllables may be a factor in achieving higher rates in SMR tasks. Speaking is a complex oromotor skill that requires the smooth coordination of several muscles of the orofacial and laryngeal system. The ability to speak is usually learned during infancy and requires a number of years before being completely mastered. Gaining insights into children’s oromotor ability from an early age is rather challenging; however, a research and clinical tool that has been widely used for this purpose in populations of different ages, starting from infants, is the oral diadochokinetic (DDK) task test . These tDDK performance is usually measured by means of the speech rate , calculaAlthough the phenomenon of AMR tasks being slower compared with SMR tasks in adults has not received much attention, previous studies have consistently reported this pattern of performance between alternating tasks and their non-alternating counterparts. Some authors have attributed those results to the overlapping gestures allowed during the execution of SMR tasks , suggestThe two hypotheses for faster rates in SMR relative to AMR tasks  relate to mechanisms of a mature motor speech encoding system, meaning that the differences between SMR and AMR might not be expected in childhood if we consider that coarticulation and other adult-like speech properties are achieved only adolescence  differences between SMR and AMR tasks would be absent in 14–16-year-old adolescents if they continue to rely on an immature motor speech encoding system; (2) SMR and AMR tasks would differ in some aspects if adolescents’ speech motor control system is nearly adult-like; and (3) 14–16-year-old adolescents would display differences between SMR and AMR tasks if their motor speech encoding system is fully mature, mirroring DDK performances in adults.To further analyze whether SMR achieving faster rates due to the chunking of the three /badego/ syllables , we condThe study involved 71 participants who were divided into three age groups: children , adolescents  and young adults . All participants were French native speakers or acquired French before the age of six, with no reported hearing, language, speech, neurologic or psychiatric disorders. Participants or their legal representatives (in the case of children) provided informed consent to be included in the study. Only children and adolescents were paid for their participation since they were involved in a different research protocol. The study was approved by the local ethics committee of the University of Geneva.The study employed the DDK tasks from the French speech assessment protocol MonPaGe ,39. The Before the production of the DDK tasks, participants were shown a visual and auditory example of the fast repetition of the syllable /ta/ to familiarize themselves with the required task. Thus, while “tatata…” was written on the screen, participants listened to the recorded example of the syllable /ta/, produced at a syllabic rate of 5.48. They were then presented with each sequence of syllables and asked to repeat it as fast and accurately as possible on a single breath for at least five seconds. In each trial, participants were first asked to read the presented sentence aloud to ensure they correctly identified and produced the target sequence . The stimuli included in the DDK task were always presented in the same order: each of the three AMR tasks /ba/, /de/ and /go/ was presented on the screen, followed by the presentation of the SMR task /badego/. Experimental runs were audio recorded separately per sequence. Participants were allowed to restart their productions whenever they failed to perform the DDK task.Audio recordings were made with a cardioid Shure SM58 microphone  and PreSonus Audiobox USB audio interface  with 24-bit resolution at a sampling rate of 48 kHz. Participants maintained a constant distance from the microphone during the recordings.The consonant and vowel segments were labeled manually in Praat  in orderFor data cleaning purposes, rates exceeding 2.5 standard deviations from each type of task’s mean per group were excluded. From the children group, one participant was excluded since all his AMR rates were considered outliers . From the group of 14-to-16-year-old adolescents, apart from one instance for the AMR rate of the syllable /ba/ not being included due to recording problems for one participant, a second /ba/ rate value from another adolescent was excluded after cleaning the data. From the adult data, one adult’s syllabic rate for the AMR syllable /de/ was also discarded following the cleaning procedure.Syllable rate data were fitted with mixed models  with RStfo) contours and intensity curves of the AMR and SMR sequences were generated for the first twelve CV repetitions using ProsodyPro  for better comparability and illustration purposes.Fundamental frequency , implemfo. To test whether SMR sequences achieve faster rates due to sequence chunking, we contrasted the fo and the intensity peaks of the syllable /go/ between AMR and SMR tasks across groups. Only the syllable /go/ was chosen here because, in French, the overall fo and intensity contours are well-known to rise on the last syllable of the sequence [Importantly, in a sequence of three /ba/ productions , only the first AMR /ba/ syllables were compared with the corresponding SMR /ba/ syllables in /badego/. The same procedure was applied to the AMR /de/ and AMR /go/ syllables  to ensure comparable values across the same vowels, especially for the sequence .The distribution of the first two formant frequencies (F1 and F2) was measured in the AMR and SMR sequences with custom Praat scripts using linear predictive coding (LPC) with variable-order prediction filters (LPC order ranging from 12 to 16) to take into account the different vocal tract sizes of children, adolescents and adults. Specifically, we calculated a compactness index between F1 and F2 for the /a/ vowels in the AMR and SMR tasks, which indicates the degree to which the F1–F2 space of one vowel moves towards another vowel category due to anticipatory coarticulation. To obtain this index, we subtracted the single F2–F1 values obtained in the middle portion of the SMR and AMR vowels /a/. The other SMR vowels  were not statistically tested.Raw data are available in the Mean AMR and SMR syllabic rates in syllables per second are shown for the three groups in p < 0.001) and of group  = 20.85; p < 0.001) as well as an interaction between the two variables  = 8.30; p < 0.001). Interaction contrasts, examined using the package “emmeans” [p = 0.11, β = −0.19, SE = 0.12), whereas a significant difference between the two types of DDK tasks was found for adolescents (t(210) = −2.99; p < 0.01, β = −0.37, SE = 0.12) and adults (t(210) = −7.29; p < 0.001, β = −0.84, SE = 0.12).The linear mixed model (Model: lmer(Rate~(task*group) + (1|subject), data = data, REML = FALSE) indicated a main effect of type of task  = 46.61; Given those results, and especially considering the smaller estimate value for adolescents as compared with that of adults, we investigated whether differences between SMR and AMR in adolescents were driven by some  of the AMR syllables. Thus, separate models for those two age groups were performed by setting the type of sequence  as fixed factor. Since we were still interested in the contrast between SMR and the three AMR syllables, /badego/ was always set as the intercept in contrasts.AdolescentsMean syllabic rates per type of sequence in adolescents are presented in p < 0.001). Contrasts revealed significantly faster syllabic rates for the SMR task as compared with the AMR syllables /de/ (t(64.06) = −3.12; p < 0.003, β = −0.30, SE = 0.09) and /go/ (t(64.06) = −6.43; p < 0.001, β = −0.62, SE = 0.09). The comparison between the SMR syllable and the AMR syllable /ba/ did not reach significance (p > 0.05).The linear mixed model on the 22 adolescents yielded a main effect of type of sequence  = 14.95; AdultsAdults’ mean syllabic rates per type of sequence are shown in p < 0.001). Contrasts revealed significantly faster syllabic rates for the SMR task as compared with all the AMR syllables: /ba/ (t(73.76) = −4.44; p < 0.001, β = −0.65, SE = 0.15), /de/ (t(73.88) = −5.15; p < 0.003, β = −0.77, SE = 0.15) and /go/ (t(73.76) = −7.42; p < 0.001, β = −1.09, SE = 0.15).The linear mixed model on the adult data indicated a main effect of type of sequence  = 19.34; Twelve speakers  were excluded from the acoustic analyses due to noise in the audio signal.fo)Fundamental frequency  + (1|Subject), data = data, REML = FALSE), revealing a main effect of group  = 18.99; p < 0.001), with children showing higher fo values as compared with both adolescents and adults, whereas no main effect of task  = 0.47; p = 0.49) and no interaction between the two factors was found  = 2.01; p = 0.13). These results suggest no difference in fo between AMR and SMR tasks for children, adolescents and adults.Intensityp = 0.97) or task  = 1.18; p = 0.27). The interaction between group and task was neither significant  = 0.23; p = 0.79). Therefore, the intensity results show no difference between AMR and SMR tasks for children, adolescents and adults, which is consistent with the fo findings reported above.Mixed models (Model: lmer(Intensity~(DDK_task*group) + (1|Subject), data = data, REML = FALSE) yielded no main effect of group  = 0.02; The F2–F1 space for the SMR and AMR /a/ vowels is shown in p < 0.001), with children having higher values compared with adolescents and adults, as well as a main effect of task  = 831.01; p < 0.001), indicating more F2/F1 distance for the SMR vowel /a/. The interaction between group and task similarly reached significance  = 6.93; p < 0.001). Relevant contrasts showed that the F2–F1 distance of the SMR vowel /a/ was significantly increased in the three groups (children: t(888) = −13.15, p < 0.001, β = −261, SE = 19.8; adolescents: t(888) = −16.41, p < 0.001, β = −308, SE = 18.8; adults: t(888) = −20.97, p < 0.001, β = −358, SE = 17.1), confirming gestural overlap for SMR sequences in children, adolescents and adults. However, by visual inspection of Mixed models on the F2−F1 distance of the AMR and SMR vowel /a/ (Model: lmer(F2F1~(DDK_task*group) + (1|Subject), data = data, REML = FALSE) revealed a main effect of group  = 9.01; fo and the resulting sparser distribution of the harmonics (which makes estimations for children a more error-prone process), a whole-spectrum measure of coarticulation was calculated. This was obtained by the Euclidean distance between average Mel-frequency spectral vectors of the /a/ vowels from the SMR and AMR tasks. Mixed models were used to statistically test the Euclidean distance between the SMR and the AMR /a/ vowels across groups (Model: lmer(EDistance~(group) + (1|Subject), data = data, REML = FALSE). The results revealed a main effect of group  = 4.21; p < 0.03), with children obtaining lower values compared with adolescents (t(59) = 2.11; p = 0.04, β = 4.81, SE = 2.27) and with adults (t(59) = 2.82; p < 0.01, β = 6.15, SE = 2.18). No differences between adolescents and adults were found (t(59) = −0.63; p = 0.53, β = −1.34, SE = 2.11). These results . Thus, contrasting SMR and AMR rates across the three age groups revealed that SMR and AMR tasks follow different performance patterns from childhood to adulthood.Producing repetitions of the same syllable has been previously suggested to differ from the repetitive production of changing syllables . ExperimThe present results clearly show that DDK tasks are characterized by a gradual distinction between SMR and AMR tasks over time, thus also confirming that DDK tasks are a heterogeneous class of motor behavior . To bettThe second hypothesis explaining the faster production of SMR sequences relies on higher gestural overlap across SMR syllables . This hyFinally, although this study adopted an acoustic perspective for investigating why SMR tasks achieve faster rates than their non-alternating counterparts, it is important to acknowledge that higher-level mechanisms might also play a role in preventing gestural overlap across repeated syllables . For instance, motor programming inhibition  may playAlthough this study included three different groups, testing the SMR–AMR performance patterns in other intermediate ages, especially in speakers from 9 to 12 years of age, is needed in future studies. Including intermediate groups would allow determination of whether the changes observed in 14-to-16-year-old adolescents follow a linear and progressive development from childhood or if a crucial switching change can be identified at a particular age. Inclusion of larger sample sizes is also encouraged in future investigations to strengthen the statistical findings, particularly in younger groups where larger between-participant variability is generally observed.The results of the present study bring important insights into the development of oromotor skills throughout a speech-like task that is easily used across different age groups. We were able to show that the production of SMR sequences becomes faster than that of AMR sequences only in mid-adolescence and that this finding seems to be related, at least partially, to greater gestural overlap across alternating syllables. A developmental pattern of intra-gestural synergies reaching adult-like patterns only in adolescence has been previously reported for speech stimuli in acoustic and articulatory studies on speech production . The faIn conclusion, the production of SMR and AMR sequences showed similar rates in children, whereas faster SMR rates were displayed in adolescents and adults as compared with AMR rates. Children achieving similar rates for the two DDK tasks seems to be due, at least partially, to a lower degree of coarticulation in the production of sequences of different syllables. The finding that faster SMR sequences are only attainable in mid-adolescence aligns with research on speech production, implying that DDK tasks could potentially provide valuable information on the state of speech motor control during development.


Strongyloides stercoralis is a soil-transmitted helminth that is mainly found in the tropical and subtropical regions and affects approximately 600 million people globally. The medical importance of strongyloidiasis lies in its capacity to remain asymptomatic and chronically unnoticed until the host is immunocompromised. Additionally, in severe strongyloidiasis, hyperinfection syndrome and larva dissemination to various organs can occur. Parasitological techniques such as Baermann-Moraes and agar plate culture to detect larvae in stool samples are the current gold standard. However, the sensitivity might be inadequate, especially with reduced worm burden. Complementing parasitological techniques, immunological techniques including immunoblot and immunosorbent assays are employed, with higher sensitivity. However, cross-reactivity to other parasites may occur, hampering the assay’s specificity. Recently, advances in molecular techniques such as polymerase chain reaction and next-generation sequencing technology have provided the opportunity to detect parasite DNA in stool, blood, and environmental samples. Molecular techniques, known for their high sensitivity and specificity, have the potential to circumvent some of the challenges associated with chronicity and intermittent larval output for increased detection. Here, as S. stercoralis was recently included by the World Health Organization as another soil-transmitted helminth targeted for control from 2021 to 2030, we aimed to present a review of the current molecular techniques for detecting and diagnosing S. stercoralis in a bid to consolidate the molecular studies that have been performed. Upcoming molecular trends, especially next-generation sequencing technologies, are also discussed to increase the awareness of its potential for diagnosis and detection. Improved and novel detection methods can aid in making accurate and informed choices, especially in this era where infectious and non-infectious diseases are increasingly commonplace. Strongyloides stercoralis, a soil-transmitted helminth (STH), is responsible for human strongyloidiasis, which is estimated to affect approximately 600 million people globally [Strongyloides stercoralis infection in humans ranges from asymptomatic light infections to chronic symptomatic infections. Severe strongyloidiasis can occur as hyperinfection syndrome (increased parasite burden resulting in high parasite load) and/or disseminated strongyloidiasis . Like a silent assassin, S. stercoralis infection can remain asymptomatic and chronically unnoticed until the host is immunocompromised [S. stercoralis to replicate itself in the human host allows for cycles of autoinfection, where the larva attains infectivity without leaving the host [globally –3. Stronglobally . Strongypromised , 6. Hypepromised , 8. Morethe host .S. stercoralis, partly due to asymptomatic infections and inadequately sensitive methods for detection and diagnosis [S. stercoralis larvae are usually released in stool samples instead. Moreover, in asymptomatic infections where the larval output is low and intermittent, the sensitivity of stool examination may be compromised [S. stercoralis detection include immunological and molecular methods, which have been dubbed a more sensitive alternative to complement diagnosis. The current molecular methods include conventional polymerase chain reaction (PCR) and quantitative PCR (qPCR), which are widely used for the molecular detection and identification of parasitic helminths [S. stercoralis diagnosis and detection remains subjective because of the differing sensitivities reported.Currently, there is consensus regarding the underestimation of the actual prevalence rate of iagnosis . In contpromised . Other melminths –12. HoweS. stercoralis with the other STHs targeted for control from 2021 to 2030 [S. stercoralis into a WHO control program includes gaining knowledge of the epidemiology of S. stercoralis, conducting field evaluations and pilot projects, and finding a suitable standard diagnostic tool for detection and diagnosis [S. stercoralis as a target for control, the importance of a sensitive and accurate technique for molecular diagnosis is crucial.Recently, the World Health Organization (WHO) included  to 2030 . Incorpoiagnosis . Due to S. stercoralis. Additionally, upcoming molecular trends, especially next-generation sequencing technologies, are discussed in this context to increase awareness of their potential for diagnosis and detection.In this study, to consolidate the molecular studies that have been conducted and to assist stakeholders in the WHO’s direction, we present an up-to-date review of the current molecular techniques used for detection and diagnosis of S. stercoralis larvae in fecal samples under microscopes [S. stercoralis eggs are not usually found; thus, parasitological techniques like the simple smear or Kato-Katz are not suitable. More appropriate parasitological methods for larval detection include the Baermann-Mores and agar plate culture (APC) [S. stercoralis does not indicate the unequivocal absence of infection [S. stercoralis detection in a combination of Baermann-Moraes and APC [Currently, parasitological techniques are the gold standard for detecting roscopes . Comparere (APC) –17. The nfection . Also, mnfection , 18. Kno and APC . HoweverS. stercoralis detection and diagnosis. They are commonly used as a benchmark to compare the efficacy of immunological and molecular techniques [Despite the low sensitivity, parasitological techniques remain the go-to method for chniques , 21. AltS. stercoralis diagnosis and present certain advantages over parasitological methods [Immunological techniques, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT), and western blot, have been used as alternatives for  methods . Various methods , 22, 23.S. stercoralis, which can be a limiting factor in areas where strongyloidiasis is endemic [The sensitivity of five immunological tests  was compared by Bisoffi et al. (2014), and their results revealed that the sensitivity among the tests ranged from 75.4% to 93.9%, with the IFAT test being the most sensitive . However endemic , 26. Mor endemic . Current endemic , 28–30.  endemic . In addi endemic , 33. In  endemic . AlthougS. stercoralis diagnosis and identification, with their potential for increased sensitivity and specificity [S. stercoralis detection. Of the 24 studies reviewed, the sensitivity ranged from 15 to 100%, while specificity ranged from 76.7% to 100%, with different studies utilizing parasitological or immunological techniques, or both as references. The majority of studies conducted used fecal samples, while three studies used urine samples for the detection of S. stercoralis DNA. The most common genetic marker used was the nuclear 18S ribosomal RNA (rRNA) gene, with 16 out of 24 (66%) studies using the 18S primers and assay developed by Verweij et al. (2009) [Molecular techniques have been touted as a promising tool for cificity , 20. Tab. (2009) .Table 2SS. stercoralis in fecal samples [S. stercoralis, enhancing the utility of molecular techniques for diagnostics and detection [S. stercoralis detection in fecal samples [COI) gene, internal transcribed spacer 2 (ITS2) region, and repetitive units have been used, the 18S rRNA gene remains a popular choice for S. stercoralis detection.The assay by Verweij et al. (2009)  targets  samples . Since i samples , 35, 36.etection . Aside fetection  develope samples . The stuS. stercoralis, the accuracy was estimated to be 71.76% using parasitological techniques as the reference and 61.85% using either parasitological or immunological techniques [S. stercoralis outweigh their limitations. First, molecular detection outperforms parasitological techniques such as spontaneous sedimentation in terms of sensitivity, and studies have revealed that the sensitivity and accuracy of diagnosis increase when a combination of techniques is applied in conjunction. Hailu et al. [S. stercoralis and revealed a higher detection rate when a combination of parasitological and molecular techniques was used as compared to a single diagnostic method [S. stercoralis detection are summarized in Table Although the sensitivity range of molecular techniques varies greatly (from 15 to 100%), molecular techniques are still highly valuable as a diagnostic tool, as only five studies reported a sensitivity of < 50%. In a systematic meta-analysis of molecular diagnostic accuracy for chniques . The advu et al.  tested fc method . The advS. stercoralis molecular identification, phylogenetics, and genetic diversity. Other types of molecular-based studies performed for S. stercoralis are summarized in Table S. stercoralis prior to individual worm DNA extraction. Other types of sample include serum, cerebrospinal fluid (CSF), and bronchoalveolar lavage fluid to detect the presence of S. stercoralis DNA. The various types of genetic markers used include the nuclear 18S and 28S rRNA genes, ITS1 region, major sperm protein (MSP) gene, the mitochondrial COI, 12S and 16S rRNA genes, and repetitive elements. Although these genetic markers can be used for molecular identification and phylogenetic studies, the 18S rRNA and COI genes are highly popular. For the 18S rRNA gene, Hasegawa et al. (2009) [S. stercoralis populations [COI gene is another genetic marker used to study the population genetics and diversity of S. stercoralis in different hosts and localities [S. stercoralis through comparative molecular studies on dog and human isolates [Aside from diagnosis and detection, molecular techniques also allow the study of . (2009)  suggesteulations . With itcalities –43. Thesisolates .Table 4SS. stercoralis detection. First, parasitological, immunological, and molecular techniques are increasingly employed for screening and confirmatory testing to broaden the net cast and to increase the detection accuracy rather than relying on one approach [S. stercoralis through these three techniques [COI genes has been used to detect S. stercoralis in serum samples [S. stercoralis in CSF and bronchoalveolar lavage fluid samples from patients, showing the high sensitivity of the technique and potential for use [S. stercoralis larvae spiked in mock helminth communities and environment matrices [Researchers have recently attempted to increase the diagnostic sensitivity for approach , 44. Zuechniques , 45, 46. samples . Third,  for use –50. Addimatrices . AlthougS. stercoralis detection, the convenience of molecular detection in the field is another advantage. A loop-mediated isothermal amplification (LAMP) assay was successfully developed by Fernández-Soto et al. (2020) [S. stercoralis detection [Strongyloides hyperinfection syndrome resulting from treatment with corticosteroids for COVID-19 [Strongyloides to prevent potentially fatal scenarios, especially when the use of corticosteroids is evident [In addition to increasing the sensitivity of . (2020)  using huetection . Anotheretection . Lastly,COVID-19 –56. With evident .S. stercoralis. As each technique  has its benefits and drawbacks, none should be used as a stand-alone test for diagnosis. Molecular techniques can play a confirmatory role in diagnosis, with their ability to circumvent both the low sensitivity of parasitological techniques and the low specificity of immunological techniques. With molecular techniques advancing at an extraordinary pace, it is certainly a keystone in strongyloidiasis detection, especially in an era where infectious diseases and zoonoses are increasing in frequency.The application of molecular techniques is undoubtedly vital to determine the true prevalence and disease burden of


Molecular-scale understanding of rheological properties of small-molecular liquids and polymers is critical to optimizing their performance in practical applications such as lubrication and hydraulic fracking. We combine nonequilibrium molecular dynamics simulations with two unsupervised machine learning methods: principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE), to extract the correlation between the rheological properties and molecular structure of squalane sheared at high strain rates ( Rheological properties of small-molecular and polymeric liquids are critical to optimizing their performance in applications such as lubrication and hydraulic fracking ,2,3. Forcontacts ,5. Similcontacts .Experimental advances have enabled the design of rheometers that can now exceed 1 GPa at  ∼30 MPa ,8. Howev ∼30 MPa , but the ∼30 MPa .In the last several decades, thanks to the increased computational power and development of reliable atomistic models, nonequilibrium molecular dynamics (NEMD) simulations have emerged as a powerful tool to extract macroscopic rheological properties of liquids sheared at high strain rates that are difficult to reach in experiments ,19,20,21ss range ,17,18,22Using a united-atom model , we perf1200 MPa ,18,24. TIn a recent paper , we intrIn this paper, we build on our earlier introductory work  and exteWe note that following our earlier work , similarIn the next section, we present the molecular model and methods, discussing details not included in our earlier papers, such as the molecular force field parameters, SLLOD algorithm, and the basics of PCA and t-SNE methods. In this section, we discuss the molecular model and associated force field parameters used to describe squalane, the NEMD simulation method including the SLLOD algorithm ,38,39 usi and j. A and B denote different types of united atoms. The LJ interactions are applied to all pairs of atoms associated with different squalane molecules, and to those atom pairs that are separated by more than 3 bonds within the same molecule. In all cases, the LJ interactions are truncated and shifted at a cutoff distance of Squalane , and The angle bending potential associated with a triplet of atoms i, j, k and l are connected in sequence , and The dihedral potential is represented as a sum of cosine terms:l papers ,41.i is connected to atoms j, k and l  package . First, barostat ,44. Tempermostat ,44. Equilgorithm  with a tlgorithm ,18.i and j. Intramolecular orientation tensors are computed for atoms i and j  for atom pair p (formed by atoms i and j) is computed as [p and the unit vector associated with the Cartesian coordinate The last 1000 frames (time samples) of the molecular trajectory data in the steady-state region are used for extracting the intramolecular and intermolecular orientation tensors between atoms puted as ,18(7)Sαi, and i by other particles derived from the total potential energy, and The shear via a fictitious external force is imposed in the D method ,38,39,46al SLLOD ,38,39, gal SLLOD , p-SLLODal SLLOD ), all oflsewhere ,48. Herex direction and a velocity gradient applied in the y direction, increases the tilt factor To implement SLLOD, LAMMPS applies a triclinic box whose dimensions can be expressed asm is the number of data points associated with that variable. Often, the data for each variable is shifted to render a mean value of 0   is a wida and b) represents the correlation between them and is calculated using the formula:Covariance between any two variables  is represented with m data points:X, an C can be seen as the variance-covariance matrix for the n-dimensional data, where the diagonal elements are the variances of the variables and the off-diagonal elements are the covariances between two different variables.Consider an X such that, in the space of the new variables, the variance-covariance matrix is a diagonal matrix, i.e., its off-diagonal elements (covariances) are 0. Let P (as yet unknown) be the transformation matrix operating on X that yields the matrix represented with the new, transformed variables C is the matrix in Equation . The ratio of each eigenvalue to the summation of all eigenvalues approximately represents the weight fraction of the information in the original data that is captured by the corresponding reduced dimension .We seek a transformation of the original matrix Equation . In the matrix C :16)ECETECETX sucUnlike PCA, the stochastic neighbor embedding (SNE)  method iIn the high-dimensional space, a Gaussian distribution is centered around a data point 5 and 50 . After dThe SNE transformation tries to fit the low-dimensional distribution to the high-dimensional distribution. The fit or “mutual consistency” of these two distributions can be measured by computing the cross entropy or Kullback-Leibler divergence (KLD)  defined The original SNE often suffers from the so-called “crowding problem”, which means that well separated data in the high-dimensional space are not well separated in the low-dimensional space. This problem can be mitigated by modifying the distribution function in the low-dimensional space to the “student’s t-distribution” . Its expression and associated qj|i  are givem is the number of data points.t-distributed SNE (t-SNE) is based on SNE with the distribution function replaced by the t-distribution in the low-dimensional space. t-distribution function has a heavier tail compared to Gaussian distribution, which enables a superior mapping for the well separated data points. In general, t-SNE can provide a good data visualization by capturing correlations not picked by linear methods such as PCA. However, t-SNE has a few drawbacks . For exaWe first summarize simulation results from our earlier work on the shear viscosity of squalane at different rates and pressures relevant to the studies performed in later subsections. Next, we present the examination of the link between rheological properties and molecular structure using the intramolecular orientation tensors. This is followed by results that probe this link using the intermolecular orientation tensors.n is between 0 and 1. For low rates s papers ,18 showele model ,54, whiceriments ,18. In sOn the other hand, for high pressures ng model ,56, whicn regime ,18. EyriGiven these alternate molecular mechanisms of shear thinning, one that relies on changes in molecular order and the other that does not, an accurate assessment of changes in molecular order with increasing shear rate for different pressures becomes important to probe the origins of the observed high-rate shear thinning behavior shown in p associated with a squalane molecule. A squalane molecule has 30 united atoms, and therefore there are 435 intramolecular atom pairs. The intramolecular structure information is encoded in the associated 435 intramolecular orientation tensors. Each of these x), velocity gradient (y), and vorticity (z), respectively. A value of 0 indicates no preference to align along the axis, i.e., the atom-pair distance vector assumes a random orientation. Positive and negative values imply the extent to which the atom-pair orientation is parallel or perpendicular to the axis, with 1 and −0.5 representing perfectly parallel and perpendicular alignments, respectively.The intramolecular orientation tensor Equation ) is defiTo illustrate,  Szz2−28 a–c startIn stark contrast, the diagonal components For higher pressures, even though all the orientation tensor components associated with the 2−28 atom pair do not change significantly with increasing rate, the viscosity continues to steadily decrease. This shear thinning may result from either a change in a different intramolecular order parameter , or from an entirely different molecular mechanism . Different atom pairs might contribute differently to the molecular order, and therefore the orientation tensor information associated with not just 1, but all 435 pairs needs to be examined. We next show the results of using dimension reduction methods to analyze and visualize this high-dimensional information.We first classify the 30 united atoms of a squalane molecule into 3 types based on their positions see : end atoEach of the 435 atom pairs is associated with an intramolecular orientation tensor having 6 non-trivial components. This high-dimensional dataset of For all 6 pressures, by analyzing the eigenvectors associated with PC1 and PC2, we find that The PCA dimension reduction results for pressures ,100 MPa a,b show ,955 MPa c–f show The PCA results shown in b values (blue symbols) are clearly separated from the near-separated atom pairs characterized with small b values (red symbols).While the long backbone-backbone atom pairs associated with different strain rates are mixed together in The emergence of organization of atom pairs at higher pressures in the reduced dimensions motivates a further classification of the long backbone-backbone pairs into near pairs, which are atom pairs separated by more than 1 but fewer than 6 bonds  and 10 Å . Discretizing this distance interval with a step size of 0.1 Å, 61 intermolecular atom pairs are generated for each strain rate and pressure. An intermolecular orientation tensor p belonging to the set of these 61 pairs using Equation (d. These 61 orientation tensors encapsulate the intermolecular orientation information at each strain rate and pressure.The analysis based on the dimension reduction of the orientation tensors associated with intramolecular atom pairs does not consider effects arising from the interactions between the molecules on the viscosity of squalane. These intermolecular effects, such as the friction between a pair of molecules, can change with strain rate and pressure, thus altering the squalane viscosity. We now examine the relationship between intermolecular orientation and shear thinning. An intermolecular atom pair is defined for atoms belonging to two different squalane molecules. This atom pair is characterized with a separation distance parameter Equation , where tP increases from 0.1 MPa to 100 MPa, before plateauing for d between the intermolecular atom pair increases, the diagonal elements of the corresponding orientation tensors exhibit a non-monotonic trend. For example, d, reaching a maximum value of 0 at d and P.To illustrate, d between the atoms is increased. For small d increases, d such that for d.The PCA and t-SNE results suggest the saturation of the molecular orientation order associated with squalane sheared under different strain rates at high pressures. Motivated by this, in order to quantify the degree of evolution in the molecular orientation with shear rate for different pressures, we extract two angles, We have explored the potential of the NEMD-ML approach in probing the molecular-scale origins of the rheological properties of short-chain molecular liquids. The exploration is based on NEMD simulations of shear flow of squalane at 293 K over a broad range of strain rates Dimension reduction using PCA of intramolecular orientation tensor datasets at low pressures However, at high pressures To further explore the correlation between shear thinning and molecular structure at high pressures, the orientation tensors of intermolecular atom pairs were calculated for different separation distances within 3.9–10 Å. Dimension reduction of these intermolecular orientation tensor datasets using PCA revealed a similar contrast between the molecular structure at low pressures versus that at high pressures. For Overall, dimension reduction results showed that shear thinning of squalane at high pressures does not rely on changes in intramolecular and intermolecular order, and therefore the viscosity reduction with increasing rate is likely the result of alternate mechanisms such as thermal-activation based flow processes, which fit the macroscopic flow curves data very well. We realize that we have explored only one liquid, so our conclusions are not generalizable to other fluids. However, the NEMD-ML approach developed in this work is versatile and can be used to investigate a wide variety of fluids. Extension of the NEMD-ML approach to probe the microscopic origins of the flow properties of other small-molecular fluids and long-chain polymeric liquids will be a subject of future work.


Antimicrobial resistance is one of the greatest public health crises of our time. The natural biological process that causes microbes to become resistant to antimicrobial drugs presents a complex social challenge requiring more effective and sustainable management of the global antimicrobial commons—the common pool of effective antimicrobials. This special issue of Health Care Analysis explores the potential of two legal approaches—one long-term and one short-term—for managing the antimicrobial commons. The first article explores the lessons for antimicrobial resistance that can be learned from recent climate change agreements, and the second article explores how existing international laws can be adapted to better support global action in the short-term. Antimicrobial resistance (AMR) is one of the greatest public health crises of our time . AMR is The biological phenomenon of AMR presents a social challenge requiring a more sustainable management of the global antimicrobial commons—a common resource with global population need. A global market failure has emerged wherein a negative demand-side externality incentivizes over-use because the full social cost of consumption is not borne by users. Uncertainty and low projected returns on investment have also disincentivized the development of new antimicrobials , 17, 22.Unlike many recent global infectious disease threats like HIV/AIDS, Zika, and Ebola, AMR is not caused by a single pathogen. Rather, AMR is a threat posed by a natural biological mechanism of action: microbes, including a range of disease-causing bacteria, viruses, fungi, and parasites, can evolve to become more resistant or resilient to particular antimicrobial agents . Bacteriaccess: we need to ensure availability and affordability of antimicrobials for the millions of people each year who face life-threatening infections without them. Second is conservation: despite the need for access, we also need to ensure the sustainability of antimicrobials through disease prevention efforts, diagnostics, infection control, surveillance and appropriate prescribing. Third is innovation: we need to develop the next generation of antimicrobials to replace those that no longer work and to invent new vaccines, diagnostics, social responses, and infection control technologies to provide alternatives. These three interlinked challenges must be tackled simultaneously. Without conservation and innovation, universal access will increase resistance and deplete existing stocks of effective antimicrobials. Conservation, if pursued alone, will constrict the market for antimicrobials, restrict investment and innovation in the field, and further hinder access as well as driving resistance in low-income countries. Innovation without conservation mechanisms will quickly diminish the efficacy of new drugs and diminish the value of investments. And innovation without better access is inequitable: new life-saving technologies should not be denied to people based on geography, socio-economic status, or ability to pay.Broadly, AMR can be conceived of as three important and interlinked social challenges Fig.  4]. Fir. Fir4]. Beyond these three social challenges, AMR poses specific technical challenges that underline the need for a multifaceted approach. First, there is a significant overall risk posed by the growing global reservoir of AMR-conferring genes, which circulate among bacterial organisms , 24. SecThe global challenge posed by AMR is unique both in terms of its socio-technical complexity and longevity. Knowledge of AMR (first termed “drug fastness”) is already over a century old ; the firTackling AMR as a multi-sectoral issue depends on improving the global governance of the antimicrobial commons , 19. ResIn the following two papers, we argue that legal approaches represent the best path forward for achieving the necessary level of global coordination and collective action. International legal agreements represent the strongest formal mechanisms through which states can make commitments to each other , but reqUltimately, the effectiveness of any international legal approach to managing the global antimicrobial commons will depend on the willingness of countries to address AMR and to adopt sufficiently robust regulations that can mitigate the AMR threat , 7, 18. In the long-term, for global AMR efforts to be truly effective, the world needs an enduring international legal agreement governing this common-pool resource—a “grand bargain” tying together global efforts across sectors, countries, and time—on how we collectively manage the antimicrobial commons to balance access, conservation, and innovation. Grand bargains are complex agreements that fully address important common needs of their parties through a series of interconnected provisions. These may each be costly to an individual party but when implemented together will collectively benefit every party. A grand bargain on the global antimicrobial commons would strategically maximize the collective situation of countries facing AMR by ensuring sustained effectiveness of antimicrobials while ensuring each country’s infectious disease burden is meaningfully improved. While grand bargains are difficult to craft, they are helpful for promoting compliance with agreements over the longer-term because they are structured to ensure that each party is incentivized to uphold their obligations.But AMR also requires action in the short-term. As a second complementary option, it could be helpful to identify “legal hooks” within existing international legal agreements where relevant AMR regulations can be developed or where small tweaks can be made to the agreement that would extend its scope to cover what is needed for containing AMR.With either of these two legal approaches, developing a microbially effective, ethically fair, and politically feasible response to managing the global antimicrobial commons will require substantial effort to clearly define AMR priorities, set a global goal or target for AMR action, and develop an agreement that is sufficiently robust and widely implemented. The first article in this special issue describes the longer-term legal approach and explores the lessons for AMR that can be learned from recent climate change agreements. We recognize, however, that negotiating an international legal agreement is a massive undertaking and the threat posed by AMR necessitates substantial immediate action. Thus, the second article in this special issue explores how existing international laws can be adapted to better support global AMR action in the shorter-term. Such an approach can address pressing needs felt acutely today, including the need to strengthen AMR surveillance, fund the development of novel antimicrobials, and reduce the use of critically important antimicrobials in agriculture.