As a major metabolite of kynurenine in the oxidative metabolism of tryptophan, kynurenic acid is of considerable biological and clinical importance as an endogenous antagonist of glutamate in the central nervous system. It is most active as an antagonist at receptors sensitive to N‐methyl‐D‐aspartate (NMDA) which regulate neuronal excitability and plasticity, brain development and behaviour. It is also thought to play a causative role in hypo‐glutamatergic conditions such as schizophrenia, and a protective role in several neurodegenerative disorders, notably Huntington’s disease. An additional hypothesis, that kynurenic acid could block nicotinic receptors for acetylcholine in the central nervous system has been proposed as an alternative mechanism of action of kynurenate. However, the evidence for this alternative mechanism is highly controversial, partly because at least eight earlier studies concluded that kynurenic acid blocked NMDA receptors but not nicotinic receptors and five subsequent, independent studies designed to repeat the results have failed to do so. Many studies considered to support the alternative ‘nicotinic’ hypothesis have been based on the use of analogs of kynurenate such as 7‐chloro‐kynurenic acid, or putatively nicotinic modulators such as galantamine, but a detailed analysis of the pharmacology of these compounds suggests that the results have often been misinterpreted, especially since the pharmacology of galantamine itself has been disputed. This review examines the evidence in detail, with the conclusion that there is no confirmed, reliable evidence for an antagonist activity of kynurenic acid at nicotinic receptors. Therefore, since there is overwhelming evidence for kynurenate acting at ionotropic glutamate receptors, especially NMDAR glutamate and glycine sites, with some activity at GPR35 sites and Aryl Hydrocarbon Receptors, results with kynurenic acid should be interpreted only in terms of these confirmed sites of action. Kynurenic acid has well‐established activity as an antagonist at glutamate receptors, especially the glutamate and glycine co‐agonist binding sites, with possibly relevant actions on Aryl Hydrocarbon Receptors and the GPR35 binding site. A single proposal that it also acts on acetylcholine nicotinic receptors is analysed and discussed in the light of at least 12 failures to reproduce the activity, and a wide range of pharmacological arguments. It is concluded that there is no confirmed, reliable evidence for any effect at nicotinic acetylcholine receptors. These activities are in the same micromolar range which block NMDA receptors in brain slices examined in previous studies. In the brain slice experiments of Hilmas et al.  , were similar to those which block NMDA receptors – similar to those reported by Hilmas et al.  in slice et al. .consistent with α7‐nicotinic receptor blockade, but does not represent proof since the apparent blockade could have been indirect, or a result of reduced acetylcholine release (see below).Secondly, the reduction in EPSP size was et al. Thirdly, the cocktail of ionotropic receptor blocking agents listed above, used at relatively high concentrations to maximise the blockade of interfering receptors needed to isolate nicotinic EPSPs, could have affected the nicotinic ionotropic receptors or ion channels associated with excitability and transmitter release in an isolated, vulnerable preparation. The mixture of antagonists could also have modified functional receptor interactions which might make cells more susceptible to kynurenic acid. Atropine, for example, has been found to have some activity on nicotinic receptors .An important observation in this study was that dimethyl‐sulfoxide (DMSO), used as the solvent for kynurenic acid by Hilmas et al.  did redu  the use of derivatives of kynurenic acid which are assumed, incorrectly, to have the same sites and mechanisms of action as kynurenate itself (ii) the use of galantamine, on the assumption that it acts uniquely on nicotinic receptors and is therefore diagnostic of their involvement, but whose basic mechanism of action cannot be confirmed and is in doubt  receptor is therefore completely spurious.From this work the NMDAR antagonists most often used experimentally are 7‐chloro‐kynurenic acid (7CKA) or 5,7‐dichloro‐kynurenic acid (57diCKA) but it must be emphasised that these do not act at exactly the same sites as kynurenic acid. Both 7CKA and 57diCKA are highly selective antagonists acting only on the glycine co‐agonist site of the prominent NR1/2 subunits of NMDA receptors  and the subunit composition of the NMDARs among many other factors. No meaningful conclusions can therefore be drawn from the use of these compounds in identifying molecular sites of action as being on NMDA or non‐NMDA receptors, especially nicotinic receptors.There are, therefore, many factors operating https://en.wikipedia.org/wiki/Ligand_(biochemistry) at nicotinic receptors with several subunit compositions  the action of kynurenate, a conclusion which mirrors the comments (expanded below) that the interaction is not of true antagonism, but of ‘response cancellation’ – galantamine simply had the opposite effect to kynurenic acid, increasing the size of the IPSCs and apparently preventing the inhibitory action of kynurenate.et al., et al.,  have also been used in neurochemical studies of GABA concentrations in the rat striatum  cannot be assumed or supported in this way.A similar story applies to another NMDA antagonist, phencyclidine. McLean et al  examinedSome groups have recognised these limitations of interpretation and have attempted to strengthen their interpretation of the actions of kynurenate by showing that α7‐nicotinic receptor blockers can prevent the effects of galantamine overcoming the actions of kynurenate. However, such observations and arguments remain entirely irrelevant as they do not exclude the possibility that galantamine might be producing its effects by acting at NMDA receptors on neurons which then secondarily activate α7‐nicotinic receptors to yield the final result.et al. et al. et al. , Fibroblast Growth Factor‐2 (FGF2) and Brain‐Derived Neurotrophic Factor (BDNF) in the hippocampus (Kita This discussion has focused on a problem arising from data which have not been reproducible in at least 13 independent studies (Table


To explore the attitudes of United Kingdom (UK) medical students regarding artificial intelligence (AI), their understanding, and career intention towards radiology. We also examine the state of education relating to AI amongst this cohort.UK medical students were invited to complete an anonymous electronic survey consisting of Likert and dichotomous questions.p = 0.01) and rated more positively to the questions relating to the perceived competence in the post-graduation use of AI (p = 0.01–0.04); despite this, a large proportion of students in the taught group reported a lack of confidence and understanding required for the critical use of healthcare AI tools.Four hundred eighty-four responses were received from 19 UK medical schools. Eighty-eight percent of students believed that AI will play an important role in healthcare, and 49% reported they were less likely to consider a career in radiology due to AI. Eighty-nine percent of students believed that teaching in AI would be beneficial for their careers, and 78% agreed that students should receive training in AI as part of their medical degree. Only 45 students received any teaching on AI; none of the students received such teaching as part of their compulsory curriculum. Statistically, students that did receive teaching in AI were more likely to consider radiology (UK medical students understand the importance of AI and are keen to engage. Medical school training on AI should be expanded and improved. Realistic use cases and limitations of AI must be presented to students so they will not feel discouraged from pursuing radiology. UK medical students do not feel adequately prepared to work alongside AI, but understand the increasing importance of AI in healthcare and would like to receive teaching on the subjectA significant number of UK medical students are discounting radiology as a possible career due to AI. Students that received AI teaching were less likely to rule out a career in radiologyAlthough the small number of students that received AI teaching felt more confident in working with AI in the future compared to students that did not receive teaching, a significant number of taught students still felt inadequately prepared.The application of artificial intelligence (AI) in healthcare continues to generate increasing interest. Within radiology, there are now AI tools that employ deep learning methods such as convolutional neural networks (CNNs) which are effective at performing narrow classification tasks where large training datasets are available. High profile examples include the classification of chest radiographs based on abnormality for triage  or pathoAs the field of healthcare AI continues to expand, it is increasingly apparent that AI education for clinicians and medical students is needed. In the United Kingdom (UK), the use of novel digital methods in health has been the subject of a recent national governmental review . Of noteIn this study, we aim to understand UK medical students’ views on AI and explore if AI influences their career intentions with specific regard to radiology. We also collated information on medical students’ understanding of AI and assessed their level of confidence in working alongside AI in the future.Students from UK medical schools were invited to complete an electronic survey  designed jointly by clinicians with an interest in AI and/or medical education. Contact was made with all UK medical schools with a request to circulate the survey to all currently enrolled students. Students were also directly invited to complete the survey via social media. Students were required to enter their institutional email address at the start of the survey, and only individuals with a valid UK medical school email address were able to participate; all responses were subsequently anonymised. It was made explicitly clear to participants at the start of the survey that their responses were anonymous. Minimal risk ethical approval was obtained from the King’s College London research ethics office (KCL-MRA18/19-11127). Informed consent was confirmed with individual participants at the start of the survey.The survey design underwent several rounds of iteration, and final validation was performed with a group of 30 medical students from King’s College London who were not included in the final survey. The final survey consisted of 11 5-point Likert questions, whereby participants rated their agreement towards a presented statement relating to their current attitudes towards AI, their career intentions towards radiology, their current understanding of AI, and their confidence in using AI tools in a routine and critical manner following graduation. Dichotomous questioning was used to determine if participants received teaching on AI and if this teaching formed a compulsory part of their curriculum. The questionnaire is attached as Additional file p value of less than 0.05 was considered statistically significant.Statistical analysis was performed using R   with RStn = 432) of respondents believe that AI will play an important role in healthcare in the future, rating their responses as either strongly agree (44.2% n = 214) or agree (45% n = 218). Just under half of the respondents  reported they were less likely to consider a career in radiology due to AI, compared to those who do not feel they are less likely to consider a career in radiology due to AI . A similar number of students also believed that some specialities will be replaced by AI within their lifetime   of respondents selecting strongly agree or agree, whilst 43.4% (n = 210) selected disagree or strongly disagree, and the remaining 12.4% (n = 60) were neutral. With regard to the current limitations of AI, more students reported they had an understanding on this than those who did not; 48.3% selected strongly agree or agree to this question , whilst 30.4% selecting strongly disagree or disagree , and 21.3% (n = 103) selected neutral. More students reported that they did not feel comfortable with the nomenclature associated with AI , than those that reported otherwise   or agree (53.7% n = 260); 9.7% (n = 47) of students submitted neutral responses on this question. 1.2% of students (n = 6) disagreed with this statement. Along this line of questioning, a significant majority of students (78.1%) also agreed with the statement that all medical students should receive training in AI as part of their medical degree; 28.1% (n = 136) and 50% (n = 242) of students selected strongly agree and agree, respectively. Neutral responses were recorded by 16.1% (n = 78) of students, and only 5.6% (n = 27) of students responded negatively when asked if all students should receive training in AI  of students found that teaching in AI was useful. Students who received no teaching were less likely to consider radiology as a possible career choice in the future compared to students who received teaching in AI   received some form of teaching on AI. Students from 12 out of the 19 medical schools reported teaching opportunities relating to AI, but none of these students received such teaching as a mandatory part of the curriculum. 86.7% (n = 50) of students agreed that they would be confident in using AI tools if required, and the same number of students agreed that they would possess a basic understanding in the methods used to assess AI performance. A slightly larger number of students felt they would possess the knowledge needed to work with AI in routine clinical practice (11.3% n = 55). Students who receive training in AI recorded more positive responses to these questions, rating themselves as being more confident in the use of basic AI clinical tools if required (p = 0.04), possess a better understanding in the assessment of AI algorithm performance (p = < 0.01), and overall felt more likely to have the knowledge required to work with AI in routine clinical practice (p = < 0.01)  [N = 263) [It is now accepted that AI will likely have a profound impact in the future practice of radiology and other medical fields. This sentiment is shared by a majority of students in our cohort with 88% agreeing that AI will play an important role in healthcare. Despite convincing arguments that AI will not replace radiologists , almost N = 322) , where oN = 263)  showed tIt is worrying that a large number of students in our cohort are discounting radiology as a possible career given the acute shortage of radiologists in the UK . The worPinto dos Santos et al (2019)  identifiStudents who did receive AI training were significantly less likely to rule out radiology as a career choice, and this may be for two reasons. Firstly, students already interested in radiology as a future career are more likely to seek information on AI to provide an informed decision on their career choice. Secondly, students who do have a better understanding of AI are likely more aware of the limitations that preclude replacement of radiologists. Furthermore, it is not unreasonable to assume that some students are encouraged by the potential opportunities that AI would present in a radiological career. Our study found that students who received teaching on the topic felt more prepared in being able to work with AI tools compared to those who did not. However, despite statistical significance between the two groups on the questions relating to post-graduation readiness on working with AI, the majority of students in the taught group still reported that they would lack both the confidence and knowledge to employ AI in a critical manner. Although most students in the taught group felt that the teaching they received is useful, our results suggest the sporadic teaching that exist within UK medical schools do not adequately prepare medical students for the impending digital revolution of healthcare. It is now accepted that AI will likely have a profound impact in the future practice of radiology and other medical fields, and it is thus inevitable that AI and other digital tools will be incorporated into clinical practice, regardless of speciality. It would be a failure if we do not seize on this opportunity to better equip our future physicians with the adequate knowledge. It is our belief that the physicians of tomorrow must possess the means to employ digital tools, including AI, in a manner that is akin to rational evidence-based medication use.Additional file 1. Questionnaire on current attitudes and understanding on artificial intelligence of medical students in the United Kingdom.


Congenital disorders of glycosylation (CDG) are a rapidly expanding group of rare genetic defects in glycosylation. In a novel CDG subgroup of vacuolar‐ATPase (V‐ATPase) assembly defects, various degrees of hepatic injury have been described, including end‐stage liver disease. However, the CDG diagnostic workflow can be complex as liver disease per se may be associated with abnormal glycosylation. Therefore, we collected serum samples of patients with a wide range of liver pathology to study the performance and yield of two CDG screening methods. Our aim was to identify glycosylation patterns that could help to differentiate between primary and secondary glycosylation defects in liver disease. To this end, we analyzed serum samples of 1042 adult liver disease patients. This cohort consisted of 567 liver transplant candidates and 475 chronic liver disease patients. Our workflow consisted of screening for abnormal glycosylation by transferrin isoelectric focusing (tIEF), followed by in‐depth analysis of the abnormal samples with quadruple time‐of‐flight mass spectrometry (QTOF‐MS). Screening with tIEF resulted in identification of 247 (26%) abnormal samples. QTOF‐MS analysis of 110 of those did not reveal glycosylation abnormalities comparable with those seen in V‐ATPase assembly factor defects. However, two patients presented with isolated sialylation deficiency. Fucosylation was significantly increased in liver transplant candidates compared to healthy controls and patients with chronic liver disease. In conclusion, a significant percentage of patients with liver disease presented with abnormal CDG screening results. However, the glycosylation pattern was not indicative for a V‐ATPase assembly factor defect. Advanced glycoanalytical techniques assist in the dissection of secondary and primary glycosylation defects.  Most CDG share a multisystem phenotype with dysmorphic features, failure to thrive, and neurological symptoms.In recent years, a novel subgroup of CDG patients has emerged that presents predominantly with a hepatic phenotype.Liver cirrhosis develops as a response to chronic liver injury. The central pathological event in cirrhosis is deposition of extracellular matrix increasing hepatic flow resistance with ensuing hepatocyte dysfunction.Abnormal glycosylation is a known phenomenon in chronic liver disease and can be used to discriminate between different fibrosis stages and cirrhosis and provides an interesting and noninvasive alternative for liver biopsy.Traditionally, new CDG patients are identified through isoelectric focusing of TF (tIEF).The primary aim of this study was to identify glycosylation patterns that could help to differentiate between primary and secondary glycosylation defects in liver disease within a cohort of adult patients with end‐stage liver disease. As a secondary aim, we studied to what extent liver disease affects CDG screening.22.1We collected 1042 samples from patients with an established chronic liver disease from four Dutch tertiary referral hospitals. Samples were collected in the period 1993 to 2013. We specifically targeted ESLD patients who were evaluated and waitlisted for LTx. These samples were provided by Erasmus MC in Rotterdam (n = 264), LUMC in Leiden (n = 142), and UMCG in Groningen (n = 155). All samples were drawn and aliquoted preceding LTx.Chronic liver disease samples (n = 410), without ESLD, were provided by the Radboudumc in Nijmegen and by the LUMC . These patients were seen at the outpatient clinic for diagnosis and treatment of a range of liver diseases. Patients with known viral hepatitis  were excluded from analysis as we hypothesized that the presence of underlying CDG would be unlikely in this patient population. Samples from healthy controls (n = 40) were obtained from the local bloodbank. All samples were stored at −80°C until analysis.www.federa.org). All experiments were performed in accordance with the guidelines and regulations of the Ethics Committee of the Radboudumc. Approval was documented in case file 2018‐5012.Material was collected in agreement with the Dutch code of conduct for responsible use of human tissue . We selected peaks corresponding to a known TF isoform with an abundance of >1000. The relative abundance of these peaks was calculated based on their percentage relative to total abundance. Figure 2.32.3.12 anti‐TF antibody  and visualized with Coomassie blue. Data analysis was performed with Image Quant Software .All samples were analyzed with tIEF to determine abnormal sialylation of TF. tIEF was performed as described before.TF polymorphisms were recognized by doubled bands for all isoforms. For confirmation, neuraminidase treatment, was applied on samples with ambiguous isoelectric patterns, as described before.2.3.2Mass spectrometry analysis was performed as described before.2.4U test when there were two groups. P‐values were adjusted for multiple testing with the Bonferroni method.For statistical analysis SPSS Statistics v. 22  was used. Due to nonlinearity of our data we used the nonparametric Kruskall‐Wallis test for comparisons of more than two groups and the Mann‐Whitney 3A total of 1042 samples were collected, 567 samples of LTx candidates and 475 samples of patients with CLD. Figure Subsequently, QTOF‐MS was performed on 247 samples and 40 healthy controls. After QTOF‐MS analysis, 125 samples were excluded because the main peak (at 79 556 Da) was of insufficient abundance, 10 samples were excluded because of an abnormal glycation profile and 3 because of a TF polymorphism . In total, 110 patient samples and 39 healthy control samples were of sufficient quality for interpretation of the glycosylation profile. Table 3.1Out of 961 samples we analyzed using tIEF, 247 samples (26%) had hyposialylation compared to our controls. Of these samples, 175 (70%) had a solitary increased percentage of the trisialoTF isoform, 42 (17%) had an elevated percentage of monosialoTF isoform and 4 (2%) had an elevated percentage of the disialoTF isoform. Mixed combinations of elevated isoforms occurred in 26 (11%) samples Figure . None of3.2Of the 110 high quality QTOF‐MS profiles, none had a distinguishable type 1 CDG pattern. The peak associated with nonglycosylated TF (peak 75 140) was only present in one LTx patient with progressive familial intrahepatic cholestasis syndrome , one CLD patient with auto‐immune hepatitis (1.1%), and three HC . Both patients had a normal percentage of asialoTF in tIEF screening and the six patients with an elevated asialoTF fraction with tIEF analysis did not have a detectable peak with mass 75 140, corresponding to nonglycosylated transferrin. Peak 77 350, which corresponds to loss of one glycan, was present in all but five samples and was not significantly different among the groups (data not shown). We did not identify samples that showed typical glycan structural abnormalities that are seen in type 2 CDG profiles and did not identify a pattern compatible with a V‐ATPase assembly factor defect.We identified two samples (2/110 = 1.8%) with clearly elevated trisialo TF isoform abundance. The first patient in the CLD group was a female, age 55 at sampling, who was seen at the outpatient clinic for hepatic steatosis. Her profile showed an elevated trisialo TF isoform of 15.0% (median for the CLD group is 2.0%) and presence of an additional isoform . There were no signs of other glycosylation abnormalities. The second patient was a male LTx candidate diagnosed with alpha‐1 anti‐trypsin deficiency. At age of sampling, he was 28 years old. His QTOF‐MS profile showed an increase in the trisialo TF isoform of 12.4% (median for the LTx group is 1.9%) and also an increased loss of 2 sialic acids (mass 78 976 Da). For both patients, we could not detect transferrin glycoforms with missing galactose residues, as for example seen in the V‐ATPase assembly defects.3.3P < 0.001). An abnormal tIEF pattern was more frequently seen in LTx candidates compared to CLD patients .To see what the effect is of liver disease on desialylation, we first compared the tIEF screening results between LTx and CLD samples. Table Next, we aimed to gain more insight in desialylation with QTOF‐MS. QTOF‐MS provides additional detail on glycan composition. To investigate desialylation, we used the combined abundance of the trisialo and the fucosylated trisialo TF isoform. Comparison of these combined peaks among the three groups only showed a slight statistically significant difference between the LTx and CLD groups Table . However3.4P < .0001 for both). These data indicate that hyperfucosylation of TF is more pronounced in end‐stage liver disease compared to chronic liver disease and healthy controls.Hyperfucosylation of liver derived proteins is a known phenomenon in a variety of liver diseases. Therefore, we calculated the fucosylation ratio for the trisialo and pentasialo TF isoforms Figure . Fucosyl4In our retrospective analysis of a cohort of 1042 liver disease patients, we found a significant percentage of patients with altered CDG screening results by serum transferrin isoelectric‐focusing. However, we did not find clear evidence for the presence of a CDG due to V‐ATPase assembly factor defects. We found that hyperfucosylation is more pronounced in LTx patients compared to those with milder liver disease and healthy controls, thereby showing that high‐resolution mass spectrometry aids the discrimination between primary and secondary glycosylation abnormalities in CDG screening.4.1Initial screening with tIEF resulted in identification of 247 (26%), mostly mild, abnormal patterns. We did not identify a typical CDG type 1 pattern, which we anticipated, as to date there is no known type 1 CDG associated with a mild hepatic phenotype.A possible reason for this might be that prevalence is too low for detection within our cohort. The exact prevalence of the whole group of CDG is unknown, but is estimated at 0.1 to 0.5/100.000 in Europe. This number is based on recent data from the European CDG network that estimated that there is a total number of 2500 recognized CDG patients in Europe.Another reason might be that glycosylation defects of CDG patients who survive into adulthood can become milder, or even normalize, over time.We identified two patients with an abnormally high percentage of the isoform with loss of one sialic acid. The reason for this abnormal pattern remains unknown. Glycosylation defects in SLC35A1‐CDG (OMIM 603585), a defect in the CMP‐sialic acid transporter, show pure desialylation effects.Streptococcus pneumoniae‐associated hemolytic ureum syndrome.Also, the presence of bacterial sialidase in serum can lead to hyposialylation of TF. This has been described for Another option is that these patients have an as yet unrecognized type of CDG. Our study design did not allow us to further investigate this as we did not have access to fresh plasma, fibroblasts or parental DNA to perform adequate genetic analyses and functional studies.4.2Desialylation has been mostly studied in the context of alcoholic liver disease. Indeed, the carbohydrate deficient transferrin (CDT) test to identify chronic alcohol intake is based on hyposialylation of transferrin. Analysis of CDT in abstaining patients with various degrees of liver disease shows a correlation of high CDT percentages with the Child‐Pugh score.ST6GAL2 and downregulation of ST6GAL1.The pathophysiological mechanism behind alcohol‐induced hypoglycosylation is not fully elucidated. Some studies suggest a primary ER defect.Hyperfucosylation of liver‐derived proteins has been most extensively studied in hepatocellular carcinoma (HCC) patients.Hyperfucosylation of transferrin was shown to be the cause of chromatographic abnormalities in CDT testing, or so‐called di‐tri‐bridging .Somewhat older data exists on fucosylation of haptoglobin in liver disease and shows hyperfucosylation in patients with alcoholic liver disease and primary biliary cholangitis.N‐glycome with a bisecting N‐acetylglucosamine.N‐acetylglucosamine. This might be explained by protein specific glycosylation.Previous work on serum glycan analysis in liver cirrhosis identified increased hypogalactosylation and increased modification of the serum 4.3One strength of our study was that we included a wide range of liver disease patients for analysis. We took care to include patients with mild liver disease but also those with advanced chronic liver disease in need for LTx. Our efforts led to the establishment of a large sample size of over 1000 liver disease patients which adds to the robustness of the study. Additionally, we were able to screen these samples with high resolution mass spectrometry that provided in depth analysis of the intact transferrin glycoprotein, including fucosylation, loss of galactose, and the absence of complete glycans.A limitation of our study is because of its retrospective character. We had access to collected serum samples but were not in possession of fresh plasma or fibroblasts of patients to perform next generation sequencing, run a CDG‐panel, or perform functional studies. We detected two patients with an increased trisialoTF isoform. Although the cause for this elevation is unknown, we believe that the QTOF‐MS analysis ruled out abnormal glycosylation due to a V‐ATPase assembly factor defect. However, we cannot completely exclude another, possibly novel, CDG.Another limitation of our study is the high percentage of exclusion of samples resulting from low abundance of serum TF in the samples. A possible explanation could be that low serum TF is associated with cirrhosis.4.4N‐glycans suggested that hyperfucosylation was increased in a single liver disease patient but not in primary CDG.Differentiating primary from secondary glycosylation defects in patients with liver dysfunction can be challenging. Previously analysis of total plasma Our current research exposes caveats in tIEF as a primary diagnostic step in CDG screening. We show that 26% of tiEF samples were abnormal, but these samples did not show a clear type 2 CDG pattern upon QTOF‐MS analysis. An important message is that tIEF screening can be false positive because of liver dysfunction.We suggest caution when interpreting tIEF result in patients with a suspected CDG and liver disease and recommend a low‐threshold to use advanced glycoanalytic methods, preferably in an expertise center. A clear hyperfucosylated pattern is more suggestive for a secondary cause and loss of galactose more suggestive for a primary glycosylation defect. When only sialylation is decreased we suggest a repeat sample to rule out the involvement of exogeneous sialidase and a critical review of the phenotype to rule out a CDG with known hyposialylation.5Our screening study did not identify V‐ATPase assembly factor defects in a cohort of severe liver disease, but we show that end‐stage liver disease is associated with hyperfucosylation of transferrin. We confirm that regular CDG screening with tIEF can be complicated by liver disease, itself associated with mildly abnormal tIEF profiles.The authors declare no potential conflict of interest.Approval was granted by the Ethics Committee of the Radboudumc and documented in case file 2018‐5012. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000.This article does not contain any studies with human or animal subjects performed by the any of the authors.Table S1 Supporting informationClick here for additional data file.Table S2 Medians of different TF isoforms measured with QTOF‐MSClick here for additional data file.


In neonatal intensive care unit (NICU) patients with intubation status, fluoroscopic evaluation for the bowel is limited. This study was to evaluate the utility of bedside upper gastrointestinal (UGI) series with delayed radiographs (DR) for assessing duodenojejunal junction (DJJ) and small bowel passage in NICU patients with nonspecific bowel ultrasonography and contrast enema findings.We reviewed clinical and imaging data for bedside UGI with DR of NICU patients from 2014 to 2019. Five abdominal radiographs were obtained at fixed time intervals of immediately after, 1 min, 5 min, 1 h, and 2 h following the administration of 5 cc/kg isotonic water-soluble contrast agent via the nasogastric tube.n = 7) or equivocal (n = 8) at immediate or 1 min radiographs. The DJJ could not be evaluated in five from four delayed passage (including two meconium plug syndrome and one gastric volvulus) and one inadequate timing. There was only one case of intestinal malrotation, which was not detected on ultrasonography, but detected at the first UGI examination with good DJJ confidence.Twenty bedside UGI with DR were performed in 17 patients . Confidence identifying the DJJ was either good (Bedside UGI with DR can evaluate intestinal malrotation using immediate and 1 min delay and small bowel passage using 1 and 2 h delay images in NICU patients with nonspecific ultrasonographic and contrast enema findings. The majority with delayed contrast passages can have bowel pathology. Because of a small number of patients in this study, further studies with more infants are needed.The online version contains supplementary material available at 10.1186/s12887-021-02554-x. Preterm neonates are defined as babies born alive earlier than 37 weeks of gestation. An estimated 15 million babies were born preterm in 2014, and this number is rising [A fluoroscopic UGI and small bowel series is the next step for the evaluation of the neonatal UGI tract and bowel passage . UGI exaThe use of bedside UGI technique was suggested in 2014  and its Institutional Review Board of Severance Hospital approved this retrospective study  and the requirement for obtaining informed consent was waived. However, informed consent for each procedure and examinations were obtained routinely from parents. We reviewed all bedside UGI examinations performed in our hospital NICU between 2014 and 2019. We generally conduct bedside UGI examinations with delayed abdomen radiographs in NICU patients who have persistent abdominal distention without obvious causes, including volvulus, in ultrasonography or have no stool passage even after contrast enema, and exhibit unstable clinical conditions to move to the fluoroscopy rooms. This study included all bedside UGI examinations and analyzed the indications and successes/failures on completion of the study.We collected data through a review of electronic medical records from January 2014 to May 2019, including gender, gestational age at the time of birth, birth weight, age at the time of examination (weeks), indications of bedside UGI, and final diagnosis of abdominal findings. Final diagnoses were based on operative or pathologic findings, or clinical follow up results.We performed bedside UGI technique with delayed abdomen radiographs as modified from the previous report . The tecThe serial radiographs were reviewed by a pediatric radiologist with 15 years of experience in pediatric radiology. Each abdominal radiograph was assessed for DJJ position and time required to identify DJJ, passage of contrast medium, and any complication such as leakage or pneumoperitoneum.Normal DJJ position was defined as lying to the left of the left spinal pedicle, over the gastric antrum, and at the level of the duodenal bulb. We evaluated whether the DJJ was seen clearly or indistinctly. When the DJJ location was clearly identified on abdominal radiographs obtained at specific time intervals, it was considered to indicate good confidence. When the DJJ was not clearly identified, but the duodenum and jejunum were seen and the position of the DJJ could be extrapolated, it was considered equivocal. Cases in which the patient’s position was rotated but normal DJJ position was presumed were considered equivocal.2).Contrast passage and bowel transit time were objectively evaluated for the level of distal contrast at each specific time interval and subjectively assessed as normal range or passage delay. Additional findings such as volvulus or contrast leakage were also recorded. To evaluate clinical impact of bedside UGI studies, final diagnosis compared with imaging findings and management change including surgery were reviewed. Radiation dose from the radiographs were checked with tube voltage (kVp), tube current (mAs) and dose area product , gasless abdomen (n = 3), vomiting or regurgitation (n = 3), and no stool passage (n = 2). Two examinations were conducted for UGI tract motility evaluation (n = 2). The indications for follow up UGI examinations were unchanged, with follow ups performed for bowel distension in all three studies.All 20 bedside UGI examinations in 17 patients were included without any exclusions or examination failures occurring during the study period. We examined 7 boys and 10 girls. The median gestational age at the time of birth was 32 weeks . The median birth weight was 1630 g . The median age at the time of UGI examination was 5.5 weeks, with a range of 0–42 weeks. One patient underwent UGI examinations twice, at the ages of 14 and 20 weeks. Another patient underwent three examinations at 6, 15, and 42 weeks of age. All others underwent UGI studies only once. Indications for the 17 initial exams were bowel distension .Radiation dose parameters for each bedside UGI radiograph were 45–52 kVp  and 1–2 mAs with the DAP of 3–8 mGy × cmn = 8) or only mild and nonspecific bowel wall edema (n = 7) on ultrasonography. There was no examination showed evidence of intestinal malrotation or volvulus on ultrasonography images.All patients underwent bowel ultrasonography before bedside UGI study within 10 days interval. Most examinations  found no remarkable bowel pathology (n = 5), and for the other two studies the times were immediately after administration of contrast medium (n = 1) and 5 min after administration of contrast medium (n = 1). There were no cases of contrast passage disturbance or medical or surgical bowel problems during follow up among patients with normal DJJ location. The patient with intestinal malrotation underwent Ladd’s operation at the age of 40 weeks, 3 weeks after the third bedside UGI study, due to midgut volvulus.The bedside UGI study findings are summarized in Table n = 4). In the other four studies, the DJJ location was identified immediately after (n = 2) or 1 h after (n = 2) administration of contrast medium.In eight cases  the DJJ confidence was equivocal, including one study in a patient whose position was rotated. Among them, two studies were follow-up exams in a patient with known intestinal malrotation. There were no cases of contrast passage delay in this group, similar to the group with good DJJ confidence. The time to identify the DJJ was 1 min after administration of contrast medium in four cases  or ascending colon  on 1 h delay images and to distal ileum  or colorectum  on 2 h delay images in most cases. Five cases showed delayed contrast passage on 1 h delay images, and four of them showed delayed contrast passage even on 2 h delay images (passage to stomach in three cases and jejunum in one case). Finally, 3/4 of these cases of delayed passage had bowel pathology (one gastric volvulus and two meconium plug syndrome)  of the patients had underlying bowel pathology. Therefore, bedside UGI examinations with fixed time interval are useful as screening and diagnostic tests for NICU patients who have only nonspecific ultrasonographic or contrast enema findings and are difficult to move.Bedside UGI tests with delayed radiographs have several advantages. First of all, it is easy to examine patients without moving them. The low-dose abdominal radiographs routinely used in NICU patients may be conducted by taking several shots before and after the injection of contrast medium, so no special techniques are required. Tracing the duodenum and confirmation of intestinal malrotation by ultrasonography requires skill and experience on the part of the examiner, but the results of bedside UGI tests are not affected by the examiner’s experience. Moreover, in cases with gaseous bowel distention which is one of the most common indications of bowel evaluation in neonates, detection of intestinal malrotation is more difficult with ultrasonography. Ultrasonography could not detect the one case with intestinal malrotation in our study, however, it is also known that up to 29% of malrotation can have normal axis of superior mesenteric artery and vein . In addiRecording a conventional UGI series is considered to be the most important tool for the diagnosis of intestinal malrotation in neonates. For the diagnosis of malrotation in neonates and infants, conventional UGI series had the positive predictive value of 90% 47/52) with 4% (4/112) false negative rate in surgically confirmed cases /52 with , and it For the patient with malrotation observed in our study, we repeated bedside UGI examinations three times, and the time of DJJ location and diagnostic confidence were different at each of these examinations: 1 min at the first exam with definite malrotation, 1 h at the second exam with equivocal confidence due to rotated position, and immediately after at the third exam in the patient with equivocal DJJ location due to gaseous bowel distention. This suggests that the optimal time for the confirmation of DJJ location is variable according to the patient’s condition, and may change between observations even in the same patient. Consistently examining all patients at a single given time is likely to cause clinicians to miss the DJJ location. Therefore, several overlapping examinations with fixed time intervals, as used in this study, are mandatory. In addition, because the study is performed at bedside, not in the fluoroscopic room, confirmation of no rotation of patients and exact timing of radiographs is important to interpret bedside UGI study.On the evaluation of contrast passage delay, there were two cases of meconium plug syndrome and one case of gastric volvulus, resulting 75% incidence of bowel pathology in passage delay cases. Midgut volvulus can occur in patients with intestinal malrotation and UGI series show the characteristic corkscrew-like appearance with duodenal redundancy . In a prThere are some limitations in our study. First, we included only 17 patients and 20 studies, a number too small to be amenable to statistical analysis. Second, we only analyzed cases of bedside UGI series and did not compare bedside UGI series with conventional UGI series directly. However, there were no cases requiring additional study due to false negative results or unexpected emergency conditions during the study period. Third, there could be selection bias in this retrospective study, even though we did not have exclusion criteria for patient selection. Fourth, we evaluated only anteroposterior views without considerations of oblique or lateral views. If the DJJ cannot be clearly depicted on a straight anteroposterior view of the UGI series, lateral views could be helpful . If the Bedside UGI series with delayed radiographs including only five abdominal radiographs of fixed time interval  after administrating contrast medium can be useful to evaluate DJJ location, identify passage delay, and detect bowel pathology in NICU patients who have nonspecific findings on bowel ultrasonography and are at risk of adverse effects during transportation. Because this study is limited due to small number of patients, additional studies with more infants are warranted to confirm these findings.Additional file 1: Table S1. Contrast passage and the location of duodenojejunal junction (DJJ) in all studies.


Z and E isomers. The Z and E isomers have distinct photoisomerization response spectra with maxima separated by 40–80 nm, consistent with theoretical predictions for their absorption spectra. Solvation of the HI molecules in acetonitrile displaces the absorption bands to lower energy. Together, gas‐phase action spectroscopy and solution NMR and UV/Vis absorption spectroscopy represent a powerful approach for studying the intrinsic photochemical properties of HI molecular switches.Indigoid chromophores have emerged as versatile molecular photoswitches, offering efficient reversible photoisomerization upon exposure to visible light. Here we report synthesis of a new class of permanently charged hemiindigos (HIs) and characterization of photochemical properties in gas phase and solution. Gas‐phase studies, which involve exposing mobility‐selected ions in a tandem ion mobility mass spectrometer to tunable wavelength laser radiation, demonstrate that the isolated HI ions are photochromic and can be reversibly photoswitched between  Charge‐tagged hemiindigo photoswitches are demonstrated to undergo reversible Z/E photoisomerization in the gas phase, proving that photoinduced structural rearrangements do not rely on a solvent but rather reflect the intrinsic characteristics of the photoswitches. Molecular photoswitches constitute the active core of many light‐responsive molecular machines and motors,Indigoid photoswitches such as hemithioindigo (HTI)Developing effective molecular photoswitches requires a detailed understanding of their fundamental photochemical properties and underlying photoisomerization mechanisms, best provided by solid connections between experiment and theory. Although, in principle, comparisons between theory and experiment are best achieved for molecules that are free from the complicating influence of solvent or substrate, there are challenges associated with isolating and characterising the photochemical properties of molecular photoswitches in the gas phase. Therefore, theoretical predictions are often compared with measurements for molecules in solution or the solid state, which are situations where the surrounding medium plays a crucial role.Z and E isomers of three newly developed, charge‐tagged HI photoswitches 1–3 , in which the target E or Z isomer populations could be separated, and, if desired, selected and exposed to tunable radiation with separation of the product photoisomer. The experimental arrangement is depicted in Figure m and electrosprayed into the source region of the IMS. Ions were pulse‐injected through an electrostatic ion gate into the first drift region of the IMS, which was filled with N2+1 % 2‐propanol buffer gas to a total pressure of ≈6 Torr. The Z and E isomer ions were separated as they drifted through the buffer gas under the influence of an electric field due to their different collision cross sections and drift velocities, giving rise to individual peaks in the arrival time distribution (ATD). As explained in the SI, addition of the 2‐propanol dopant to the buffer gas resulted in better isomeric separation.A primary goal of the current study was to investigate the properties of the target HIs E and Z isomers for HIs 1–3. The isomeric composition of the electrosprayed solution could be altered by exposing it to visible light. Exposing the HI solution to blue light prior to electrospray ionization enriched the E isomer population, whereas exposure to green or red light enhanced the Z isomer population. These changes are clearly apparent in the ATDs shown in Figure First, we investigated the isomeric composition of the HI solutions by obtaining ATDs whereby the ions were allowed to pass unencumbered through the second ion gate. As shown in Figure eff ≈300 K), the measured ATDs represent averaged contributions from several conformers. As demonstrated in temperature‐dependent ion mobility measurements, rapid interconversion between different conformations can result in sharp ATD peaks.Z and E isomer were found to have relative energies <10 kcal/mol with respect to the corresponding most stable conformation and may be present under the prevailing experimental conditions  and indigo planes  level of theory with polarizable continuum model for the neutral, solvated Z isomer analogues of 1 (34°) and 3 (37°).Because rotations around single bonds in the alkyl chain and around the bond adjacent to the isomerizing double bond are energetically feasible at the prevailing effective temperature in the ion mobility spectrometer Teff ≈00 K, the2 buffer gas for the E isomers are 1–4 % larger than for the corresponding Z isomer for each HI species  was selected using a pulsed ion gate located halfway along the ion mobility spectrometer drift region  as shown in Figures Z→E and E→Z photoisomerization in the gas phase, in contrast to the situation observed in a recent investigation of another charge‐tagged hemithioindigo, where reversible photoisomerization was observed in solution but only Z→E photoisomerization was observed in the gas phase.Z→E PISA spectra of HIs 1, 2 and 3 occur at 450, 410, and 470 nm, respectively, whereas the corresponding E→Z PISA spectra have maxima at 505, 490, and 520 nm, respectively. The significant red shift observed for the peaks in the PISA spectra of HI 3 compared to HI 1 and 2 can be explained by the presence of the strong electron donating julolidine group, as found recently for the neutral analogue of this compound.1, 2 and 3 display clear photochromic behavior in the gas phase, with the Z isomers responding 40–80 nm to shorter wavelength compared to the E isomers, consistent with absorption spectra for the isomers in solution  level of theory using the MRCC software.1←S0 excitation wavelengths for the various low energy conformers for each HI isomer – generally, conformers with the charge tag adjacent to the donor aniline (HI 1 and 2) or julolidine (HI 3) absorb at shorter wavelengths than conformers in which the charge tag is adjacent to the indigo or distant from both the indigo and aniline. This is consistent with expectations based on the donor‐acceptor nature of the electronic transitions whereby a positive charge adjacent to the aniline (julolidine) or indigo stabilizes the S0 or S1 state, respectively.To gain insights into the electronic transitions of HIs 1←S0 transitions for the Z and E isomers of HIs 1–3 lie in the wavelength range of the measured photoisomerization response  correspond closely with the maxima of the PISA spectra  agrees with the peak photo‐response for the Z isomer of HI 2, but the peak photo‐responses for the Z isomers of HIs 1 and 3 are shifted to longer wavelength by ≈50 nm compared to the calculated values (409 and 419 nm). This may indicate that for the Z isomers of HIs 1 and 3, which have longer alkyl chains, the conformers probed in the drift tube, have, on average, the charge tag either adjacent the indigo or distant from both the indigo and aniline (julolidine), whereas for the Z isomer of HI 2 , the probed conformers are more likely to have the charge tag adjacent to the aniline. This situation appears to prevail in solution where the 1H NMR signals of the charge‐tag protons are shifted significantly upfield for the Z isomer of HI 2 compared to HIs 1 and 3, signifying its closer proximity to the aniline aromatic ring (see SI for details).Generally, the calculated vertical SZ and E isomers were separated by virtue of their different collision cross sections with N2 buffer gas allowing measurement of isomer‐specific photoisomerization responses, thereby obviating the need for deconvolution of spectra for isomeric mixtures in solution. All three HIs undergo reversible photoisomerization upon exposure to visible light, exhibiting clear photochromic behaviour in the gas phase and in solution. The gas‐phase photoisomerization action spectra are broadly consistent with calculations which capture their intrinsic molecular properties, particularly the vertical excitation wavelengths. The length of the charge‐bearing alkyl chain affects the photo‐response of the Z isomers, conceivably because it dictates the average distance between the charge tag and the donor dimethyl aniline or julolidine. Overall, the tandem IMS approach offers unique insights into the intrinsic photochemical properties of photo‐active molecules free from environment influences. Future investigations of other selected hemiindigo and hemithioindigo isomers in the gas phase should help benchmark theoretical models and build foundations for the rational design of effective molecular photoswitches.In conclusion, we have characterized three permanently charged thermally bistable HIs and reported on their photochemical properties in the gas phase and in solution. For the gas‐phase measurements, the The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Agricultural production faces a Herculean challenge to feed the increasing global population. Food production systems need to deliver more with finite land and water resources while exerting the least negative influence on the ecosystem. The unpredictability of climate change and consequent changes in pests/pathogens dynamics aggravate the enormity of the challenge. Crop improvement has made significant contributions towards food security, and breeding climate‐smart cultivars are considered the most sustainable way to accelerate food production. However, a fundamental change is needed in the conventional breeding framework in order to respond adequately to the growing food demands. Progress in genomics has provided new concepts and tools that hold promise to make plant breeding procedures more precise and efficient. For instance, reference genome assemblies in combination with germplasm sequencing delineate breeding targets that could contribute to securing future food supply. In this review, we highlight key breakthroughs in plant genome sequencing and explain how the presence of these genome resources in combination with gene editing techniques has revolutionized the procedures of trait discovery and manipulation. Adoption of new approaches such as speed breeding, genomic selection and haplotype‐based breeding could overcome several limitations of conventional breeding. We advocate that strengthening varietal release and seed distribution systems will play a more determining role in delivering genetic gains at farmer’s field. A holistic approach outlined here would be crucial to deliver steady stream of climate‐smart crop cultivars for sustainable agriculture. The current annual yield gains (≈1%) reported for major crops, that is wheat, rice, maize and soybean remain less than what is projected (≈2.4%) to reach the goal of doubling global production .A contiguous and well‐annotated genome sequence is the foundation for downstream analyses such as gene/trait discovery, genome dynamics, phylogenetic and evolutionary studies, and better cataloguing of repeat elements  using WGS strategy. However, this strategy yielded a fragmented assembly and proved costly at that time due to its reliance on Sanger chemistry ‐by‐BAC approach that relies on construction of a minimum tiling path (MTP) based on overlapping BAC clones AGI, , the Araet al.  assembleet al., et al., et al., de novo whole genome assembly created solely with short‐read technologies was strawberry (Fragaria vesca), and the authors used 454, Illumina and SOLiD platforms to decode the whole genome  leveraged the WGS strategy by dramatically improving sequencing throughput at a much reduced time and cost for genome sequencing projects  with a contig N50 of 1.5 Mb , B. oleracea (630 Mb) and Musa schizocarpa (587 Mb) showed up to 450‐fold improvement in contiguity over the existing assemblies , 3000 chickpea genome sequencing initiative (unpublished) etc. Notable in this context is recently proposed 10 000 plant genomes sequencing project (10 KP) with the aim to deliver more than 10 000 genome sequences across plants and eukaryotic microbes (https://db.cngb.org/10kp/). 10KP is a key component of EarthBioGenome project (https://www.earthbiogenome.org/) with the aim to generate sequence data for 1.5 million known eukaryotic species over a 10‐year period.Stimulated by the technological innovations, researchers are undertaking ambitious projects that intend to offer deeper insights into the genomic architectures and evolution .Availability of the reference genome sequence has stimulated sequencing of multiple accessions of a plant species to enable genome‐scale investigations. For instance, Morrell et al.  highligh et al., . Sequencde novo, which serves as a reference for the mapping of resequencing data from the remaining accessions. The super‐pangenome thus constructed offers better insights into the indispensable genome set and hence has a greater utility for crop improvement  while encompassing high diversity of a species  mapping methods suffer from limited genetic resolution besides having low throughput, being labour‐intensive and time‐consuming in nature. Presence of whole genome sequence in concert with advances in DNA sequencing technologies and computational biology has greatly empowered trait analysis and gene discovery in plants  is accompanied by sequencing of selected BC1F2 pools at relatively high coverage. This allows a quantitative trait to be analysed in a ‘near qualitative’ fashion. Using this strategy, these researchers located a plant height QTL of maize (qPH7) to a 150‐kb genomic interval harbouring a causal gene that codes for an NF‐YC transcription factor.Alternative approaches based on sequencing of selected/bulked individuals such as QTL‐seq have been widely applied for trait mapping across different crop species owing to its inherent ability to address both qualitative and quantitative traits Table . To thiset al.  have proet al., et al., et al., et al., et al., et al., With high‐throughput genotyping systems coming within grasp of even small‐scale laboratories, the type of the genetic material being employed for trait mapping studies assumes greater significance , LOC_Os11g08410 (plant height and panicle length), LOC_Os04g52479 (panicle no. per plant and spikelet number per panicle) and LOC_Os08g37890 (awn length) /MTAs along with validating the loci identified previously through QTL mapping or association studies. In soybean, GWAS on 302 sequenced genotypes identified several new MTAs that remain congruent with the previously identified QTLs controlling a range of domestication‐related traits  edits. This type of editing targets a region of the coding regions or sometimes a regulatory sequence. CRISPR/Cas9 induces a double‐strand break in the DNA and relies on the cell’s endogenous DNA repair mechanisms to religate the broken strands. While majority of DNA repair mechanisms are intrinsically accurate, it is error prone, and it is these errors that make new alleles. As already stated, these frequently result in new non‐functional alleles. However, at a low frequency they may simply cause a single non‐synonymous amino acid change. These repair mechanisms can also delete nucleotide in multiple of three, which will lead to deletions of amino acids or even peptides and these may alter the gene expression without actually creating a null allele. In a number of jurisdictions, including the United States, Australia, Japan and Argentina, these types of changes give rise to plants that are not regarded as transgenic and hence can be rapidly used in breeding programmes.It is also possible to apply gene editing such that a specific repair template is used to change the coding or regulatory sequence in a specific manner. These homology directed repair (HDR) edits can be extremely powerful tools to edit genes and create novel variants. New or different amino acids may be introduced, from a single amino acid up to hundreds of amino acids, depending on the templates used. However, there are considerable restrictions to their usage, and in many jurisdictions, the introduction of any new DNA to the host is sufficient for these to be classified as transgenic. In others, such as in the United States and Japan, they may be considered as non‐transgenic on a case‐by‐case basis.Arabidopsis and crop plants including rice  can be made in the actual gene, and hence, there are no position or dosage effects. Gene expression can be totally knocked out, which has previously been difficult using RNAi approaches, which usually lead to a diminution of gene expression but rarely to zero  or response to selection (R) can be improved by tweaking additive genetic variation (σa) or narrow sense heritability (h2), selection intensity (i) and selection accuracy (r) and length of the breeding cycle (L). In the following section, we discuss the new breeding methodologies that address different components of the breeder’s equation and improve the rate of genetic gain in a breeding programme.Genetic gains from a selection programme can be expressed in the form of breeder’s equation, that is Δet al., The paradigm ‘genotype once phenotype many times’ has dominated genetic studies for the past two decades owing to the high cost of genotyping. The increased availability of homozygous immortal genetic populations allowing replications across time and locations further fuelled this paradigm (Srivastava r) and reducing the breeding cycle length (L). GS predicts genetic merit of unobserved phenotypes from target population based on the breeding values (GEBVs) computed from genome‐wide information of a training set that has been scored phenotypically.Genomic selection (GS) improves genetic gain by enhancing selection intensity (i) and selection accuracy  could contribute to improving genetic gain. Low SRR in India despite increased availability of quality seed is due to farmers being accustomed to use >70% farm‐saved seed for raising the succeeding crop . More recently, specific‐locus amplified fragment sequencing (SLAF‐seq) technology and customized SNP array (maizeSNP3072) were optimized to support varietal identification in soybean .Seed certification being an essential step for seed quality control (QC) merits attention of both public and private agencies. Flexible systems for seed certification are warranted such that of quality declared system (QDS) adopted in countries such as Kenya and Zambia, where seed certification is licensed to private institutions  are suited more for defect elimination of mega‐varieties; however, enhancing genetic gains per unit time warrants rapid population improvement informed by genome‐wide predictions and associations (Varshney The authors declared that they have no conflict of interest.AB and RKV jointly developed the conceptual structure. AB prepared the original draft. UCJ and IDG contributed specific sections. RKV edited manuscript. All authors read and approved the final manuscript.


Although Nexplanon is one of the most effective and most utilized long‐acting reversible contraceptives in Ghana. We report a rare event of Nexplanon failure in a woman with human immunodeficiency virus (HIV) infection in rural Ghana. Although Nexplanon is one of the most effective and most utilized long‐acting reversible contraceptives in Ghana. We report a rare event of Nexplanon failure in a woman with human immunodeficiency virus (HIV) infection in rural Ghana. There are limited studies on the failure of subdermal contraceptive implants such Nexplanon in clients with HIV infection.2The client is a 37‐year‐old widow with weigh of 68kg and was diagnosed of HIV infection in 2014 and has been on the following antiretrovirals: Tenofovir (300 mg); Lamivudine (300 mg); and Efavirenz (600 mg). Diagnosis of HIV infection was made using first response rapid diagnostic HIV test kits and confirmed with OraQuick test. The diagnosis of HIV infection and regimen of treatment in this woman was based on local protocols. Her parity is six with four children alive with two infant deaths. All six gestations were carried to term, uneventful with spontaneous vaginal deliveries. She has a regular menstrual cycle of 28 days. She has no history of other chronic diseases such as diabetes, hypertension, and epilepsy. She has no history of contraceptive failure including Nexplanon. She is not on any other medications except the antiretrovirals. She also has no history of smoking or alcohol consumption.The client presented to the family planning clinic of a health facility in rural Ghana on the 6th of December 2017 and requested for Nexplanon (68 mg Etonogestrel) after informed counseling on all available contraceptive methods. Her last menstrual period was on 30th November 2017, and a urine pregnancy test was also done to rule out pregnancy. The client was eligible for Nexplanon based on the WHO Medical Eligibility Criteria for contraceptive use.On the 13th of February 2020, the client reported to the health facility with the complains of amenorrhea for six weeks, feeling of breast heaviness and dizziness. Client tested positive for pregnancy at the health facility. An obstetric ultrasound scan confirmed a single viable intrauterine gestation at six weeks and three days with no visible fetal pole yet ‐based antiretroviral while on Nexplanon. Etonogestrel is metabolized by cytochrome P450 3A4 (CYP 3A4), and EFV enhances the systemic clearance of coadministered drugs that are cytochrome CYP 3A4 substrates.Studies have reported Nexplanon failure in patients on EFV.Although Nevirapine and EFV are inducers of CYP 3A4, these drugs are part of the recommended first‐line regimen for treatment of people living with HIV including women of reproductive age according to the WHO.4This case report emphasizes the possibility of unplanned pregnancy with the concurrent use of Nexplanon and EFV‐based antiretrovirals although the WHO medical eligibility criteria permit the use of Nexplanon in women who are on EFV‐based antiretrovirals. However, further studies are needed to establish whether a causal relationship exists between EFV‐based antiretrovirals and Nexplanon failure.None declared.MTK and EAA: involved in care of the patient. MTK and PAA: prepared the manuscript. EAA: edited the manuscript. All authors: read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and its accompanying image. A copy of the written consent is available for review by the Editor‐in‐Chief of this journal.


ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one‐third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling‐centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.The endoplasmic reticulum ( The current article reviews the most up‐to‐date literature on Endoplasmic Reticulum (ER) biology and articulates this information from a signalling perspective. Not only do we cover the basic cell biology aspects of adaptive ER signalling but also provide information about the latest discoveries on ER stress targeting drugs and their potential use in the clinic. The SER and RER can exist either as interconnected or spatially separated compartments The ER is involved in many different cellular functions. It acts as a protein synthesis factory, contributes to the storage and regulation of calcium, to the synthesis and storage of lipids, and to glucose metabolism N‐glycanase  ER membrane ATP‐dependent Ca2+ pumps for cytosol‐to‐lumen transport; (b) ER luminal Ca2+‐binding chaperones for sequestering free Ca2+; and (c) ER membrane channels for the regulated release of Ca2+ into the cytosol. These mechanisms are facilitated by a tight communication between the ER and other organelles, such as the PM and the mitochondria, thereby supporting the cell needs.CaTraditionally thought as a site of protein synthesis, recent evidence has established the involvement of the ER in many different cellular functions: from novel roles in lipid metabolism to connections with cytoskeletal structures or roles in cytoplasmic streaming, our view of the ER keeps rapidly expanding, placing it increasingly as a key organelle governing the whole cellular metabolism.Conditions that disrupt ER homeostasis create a cellular state commonly referred to as ‘ER stress’. The cellular response to ER stress involves the activation of adaptive mechanisms to overcome stress and restore ER homeostasis. This response is dependent on the perturbing agent/condition and the intensity/duration of the stress 2+‐dependent apoptotic response Cell autonomous mechanisms can lead to ER perturbation and examples of this can be seen in several diseases, including cancer, neurodegenerative diseases and diabetes. The hallmarks of cancer such as genetic instability and mutations In tumours, the ER stress observed in rapidly proliferating cells is compounded by the fact that increased proliferation eventually depletes the microenvironment of nutrients and oxygen, causing local microenvironmental stress and resulting in hypoxia, starvation and acidosis, all of which cause ER stress and perturb protein, and possibly lipid synthesis 2+‐ATPase (SERCA) with compounds, such as thapsigargin and cyclopiazonic acid 2+ concentration and impairing protein folding capacity.Several small molecules that induce ER stress through a variety of mechanisms have been identified Conversely, other molecules have been found that can alleviate ER stress. These include small molecules, peptides and proteostasis regulators. The frequently used 4‐phenylbutyric acid (4‐PBA) reduces the accumulation of misfolded proteins in the ER Body temperature is crucial for the viability of metazoans; normal mammalian physiological temperatures are 36–37 °C. Deviations from this range can disrupt cellular homeostasis causing protein denaturation and/or aggregation 2+, ER oxidoreductin 1 (ERO1) and PDI Several external agents can induce intracellular reactive oxygen species (ROS) production, and when ROS production exceeds the antioxidant capacity oxidative stress negatively affects protein synthesis and ER homeostasis In response to ER stress, cells trigger an adaptive signalling pathway called the unfolded protein response (UPR), which acts to help cells to cope with the stress by attenuating protein synthesis, clearing the unfolded/misfolded proteins and increasing the capacity of the ER to fold proteins.GRP78) (BiP)], keeping them in an inactive state The UPR is a cellular stress response originating in the ER and is predominantly controlled by three major sensors: inositol requiring enzyme 1 (IRE1), protein kinase RNA‐activated (PKR)‐like ER kinase (PERK) and activating transcription factor 6 (ATF6). The ER luminal domains of all three ER stress sensors are normally bound by the ER resident chaperone, heat shock protein A5  pathways of apoptosis. Both pathways trigger activation of caspase proteases that dismantle the cell, and all of the three branches of the UPR are involved in apoptosis. In the extrinsic pathway, the activation of DRs on the PM leads to the recruitment of caspases to the DRs and their proximity‐induced trans‐autoactivation. Intrinsic apoptosis involves the release of cytochrome Although IRE1–XBP1s signalling is mainly prosurvival, IRE1 can promote apoptosis. Activated IRE1 can interact directly with TRAF2, leading to the activation of apoptosis signal‐regulating kinase 1 (ASK1) and its downstream targets c‐Jun NH2‐terminal kinase (JNK) and p38 MAPK Interestingly, recent studies indicate a role for miRNAs in the induction of apoptosis following prolonged ER stress. For example, miRNA29a which is induced during ER stress via ATF4 results in the downregulation of antiapoptotic Bcl‐2 family protein Mcl‐1, and thus promotes apoptosis in vitro and in vivo evidence have highlighted that the activation of the PERK arm of ER stress evoked in response to selected of anticancer therapies , RIPK3 and mixed lineage kinase domain‐like (MLKL) protein and has been linked to ER stress. In an 2+ release from the ER lumen through the IP3R can relieve mTOR inhibition on the ULK1 complex Endoplasmic reticulum stress has also been connected to autophagic cell death. Autophagy not only promotes cell survival, but can also mediate nonapoptotic cell death under experimental conditions when apoptosis is blocked, or in response to treatments that specifically trigger caspase‐independent autophagic cell death Endoplasmic reticulum stress causes morphological changes in cellular models. Experiments to date have largely focused on the morphologies associated with apoptotic and autophagic cell death resulting from UPR activation. UPR‐regulated flattening and rounding of cells, indicative of cell death, has been observed in many model systems, with traditional caspase‐dependent apoptosis being responsible N‐cadherin in models of colorectal, breast and pulmonary fibrosis An intensively studied consequence of ER stress is the epithelial to mesenchymal transition (EMT) and its role in cancer invasion and metastasis. EMT is an essential component of tissue repair following wounding, allowing for the migration of new healthy cells into any lesions that have occurred. Morphological changes indicative of EMT have been observed in multiple cell models under physiologically relevant stress (e.g. hypoxia) and pharmacological induction of ER stress −/− murine models subjected to intermittent water deprivation, similar downstream effects were observed, but signalling pathways were not investigated The tissues and cells of the endocrine system responsible for hormone production and extracellular signalling often have a high protein load, resulting in ER stress and activation of the UPR. OASIS (CREB3L1) and ATF6α have been shown to regulate arginine vasopressin (AVP), a potent vasoconstrictor, in murine and rat models It has been established that ER stress signalling is important in interorganelle and intercellular interactions. It therefore comes as no surprise that it forms a significant network of interactions upon which normal physiology is based. This is not only the case in humans, but is also conserved throughout species and has been an important fact in the design of experimental model organisms to further study ER stress signalling and it role in physiology and disease.Caenorhabditis elegans and Drosophila melanogaster is IRE1 in vitro or naturally, undergo a multitude of physical, biochemical and cellular stresses involving epigenetic changes as well as a disproportional increase in protein synthesis load that affect cell differentiation, proliferation and growth.C. elegans it has been postulated that the IRE1‐XBP1 axis as well as the PERK pathway are responsible for the maintenance of cellular homeostasis during larval development Xenopus embryos, where BiP morpholino knockdown not only blocked pronephros formation but also attenuated retinoic acid signalling, impacting markers such as the Lim homeobox protein The UPR as the major conduit of ER stress regulation has been extensively studied in developmental biology in the majority of organisms commonly used in translational research. The use of multiple models has been important in discerning the variable ER stress signalling between species, as demonstrated by the discovery that protein quality control in mammals is critically dependent on ATF6 while the major player in Many cell types experience a high protein load during various stages of differentiation and maturation, resulting in ER stress. In several cases, morphological changes required for the final function of the cell would not be possible without transient activation of the UPR's cytoprotective mechanisms. Deletion of PERK in murine models results in loss of pancreatic β cell architecture but not in cell death, and was accompanied by an increase in β cell proliferation. This morphological change results in a diabetes mellitus‐like pathology and is not a result of increased cell death as previously proposed The ER is a site of significant metabolic regulation. The UPR plays a major role in the regulation of glycolysis and it was recently shown that IRE1 mediates a metabolic decrease upon glucose shortage in neurons, suggesting an important role for the UPR as an adaptive response mechanism in relation to energy metabolism The ER is heavily involved in lipid homeostasis. Characteristically, hepatocytes are enriched in SER, because in addition to protein synthesis, these cells also synthesize bile acids, cholesterol and phospholipids. XBP1 ablation in murine liver results in hypolipidaemia due to feedback activation of IRE1 caused by the lack of XBP1. Activated IRE1 induces the degradation of mRNAs of a cohort of lipid metabolism genes via RIDD, demonstrating the critical role of IRE1–XBP1 signalling in lipid metabolism and suggesting that targeting XBP1 may be a viable approach to the treatment of dyslipidaemias INS1, PC1 and SYP. These effects can be observed in cases of XBP1 deficiency and in cases of extensive UPR activation, highlighting the divergent effects of IRE1 RNase activity The UPR is also involved in regulating glucose metabolism. Initial murine studies suggested the PERK–eIF2α arm was responsible for impaired insulin signalling due to knock out effects on beta cells during development. Further studies have since shown that IRE1 RIDD activity is responsible for a reduction in the mRNA of proinsulin processing proteins, including The UPR is also described to be involved in amino acid metabolism. It was recently described that ATF4 mediates increased amino acid uptake upon glutamine deprivation Several small molecules have been reported to modulate (activate or inhibit) one or more arms of the UPR. Importantly, these molecules have shown promising beneficial effects in diverse human diseases Table . X‐ray cIRE1 signalling information along with CHOP/Gal4‐Luc cells and UPRE‐Luc engineered cells were used to screen large chemical libraries in high throughput screening assays for discovery of pathway‐selective modulators of IRE1 apo dP‐IRE1* (PDB 5HGI), as a back‐to‐back dimer. Notably, the DFG motif (Asp711‐Phe712‐Gly713) faces into the active site (DFG‐in), with helix‐αC‐in conformation. In contrast, human IRE1 bound to KIRA compound 33 (PDB: 4U6R) shows an inactive kinase conformation, with DFG‐in and helix‐αC‐out conformation. The inactive conformation is incompatible with back‐to‐back dimer formation due to the displaced helix‐αC IRE1 modulators have been discovered primarily by traditional drug discovery methods, identifying inhibitors specific to the kinase or RNase domain  or preventing the generation of glycoproteins, and hence, the induction of ER stress through calcium imbalance or misfolded protein accumulation. They were touted as potential antitumour therapies as they could potentially induce tumour cell death through ER stress overactivation. However, none of these compounds were used in the clinic due to their lack of specificity and high toxicity. It has been reported though that a prodrug analogue of thapsigargin, mipsagargin, did display acceptable tolerability and favourable pharmacokinetic profiles in patients with solid tumours In this section, we review recent preclinical and clinical studies in which UPR components were used as disease biomarkers or as therapeutic targets Fig. . As alreChanges in UPR and ER stress markers in blood or tissue biopsy samples can be indicative of disease state and could be/are utilized as valuable biomarkers for different human pathologies. For instance, BiP has strong immunological reactivity when released into the extracellular environment Beyond their use as biomarkers, ER stress signalling components also represent relevant therapeutic targets. BiP was recently recognized as a universal therapeutic target for human diseases such as cancer and bacterial/viral infections ER stress‐induced transcription factors can also represent relevant targets. Thus, XBP1s has been one of the main targets for drug discovery and gene therapy Listeria monocytogenes in a mouse model ER stress targets are also strong candidates for immunotherapy and vaccines development, a good example of which is the production of chaperone protein‐based cancer vaccines termed chaperone‐rich cell lysate (CRCL) As discussed above, understanding and characterizing the UPR has provided several potential targets to develop new therapeutics for various diseases, with an encouraging increase in the number of clinical trials based on ER stress pathway targets or associated drugs. Several of these trials  have focused on diabetes mellitus. A trial testing TUDCA and 4‐PBA for the treatment of high lipid levels or insulin resistance was conducted by the Washington University School of Medicine; however, although this study was completed in 2014, the findings are not available yet. The results from the first completed human trial using BiP for rheumatoid arthritis are described in Box Box 1et al. In 2006, Brownlie The ER has evolved in our knowledge from a key player in proteostasis and the secretory pathway to a cornerstone of metabolic functions. Such wealth of information has allowed the identification of numerous mechanisms for fine‐tuning ER signalling, as well as motivated the need for their better characterization towards relevant health‐related applications. This drive to further ER knowledge has also led to the identification of emerging roles for the ER in physiology and disease. In particular, it appears an indispensable tool for cellular communication that reaches beyond the intracellular space. The concept of transmissible ER stress illustrates the far‐reaching control that ER signalling exerts in interorgan communication affecting disease pathogenesis and normal physiology Our increasing knowledge of ER signalling mechanisms presents opportunities to exploit the resulting applications on multiple fronts, including bioengineering and health, concepts that may routinely overlap. For example, boosting ER protein production capacity may be applied to cell engineering to increase biologic therapy production. This will drive down costs of biologics, helping demand to be met and leading to more widely available medications, thus having a significant effect on public health. Population‐wide consequences of ER modulation may not be restricted to the production of biologic therapies as its applications could also contribute to bioengineering approaches for crop or livestock improvement.in silico strategies supplies researchers with a wealth of tools to study pathophysiology, from structure to function in multiple in vivo and in vitro models, producing robust results to be put forward for clinical scrutiny while all the while observing both the safeguards of the declaration of Helsinki and ethics on animal experimentation. Our deeper understanding of the ER and its major homeostatic regulator, the UPR response, is introducing an individualized molecular approach to health management at a preventative, diagnostic and therapeutic level and, uncovering the genetic architecture underlying the ER stress response could significantly influence future therapeutic strategies in patients.A thorough understanding of the ER stress response and its role in physiology and pathophysiology can be applied to develop new ER stress targeted therapies and stratifying patients into cohorts suitable for ER‐targeted therapies. Considering the enormity of attrition rates of novel therapeutic discovery in an ever‐tightening financial climate, there is an urgent need for new therapeutic targets as well as precision tools that target and guide innovation to specific patient pools. ER stress signalling may provide such tools. Not only is it central to life itself but it is involved in a wide array of clinical presentations. Moreover, its effect on heterogeneous presentations within the same diseases makes it an attractive target for translational precision medicine. Of course, when undertaking medical research or trying to solve a biomolecular functional mystery one cannot look past the logistical aspect of the task ahead. The conserved metazoan nature of ER stress signalling combined with the emergence of high throughput and


Generally, the postoperative examination of lymph nodes (LNs) is based on a microscopic examination of one hematoxylin and eosin (HE)‐stained slide; however, an examination of only one part of the LN might lead to incorrect staging of the tumor due to tissue allocation bias. Although multilevel sectioning and the use of immunohistochemistry (IHC) have improved the detection of micrometastases in LNs, this approach is laborious, time‐consuming, and costly. A novel molecular technique for the detection of LN metastases of tumors, called one‐step nucleic acid amplification (OSNA), is a rapid and semi‐quantitative examination quantifying the number of cytokeratin 19 (CK‐19) mRNA copies derived from a tumor. OSNA is already in clinical use for the diagnosis of LN metastasis in breast cancer patients; however, the use of OSNA is under investigation with promising results for colorectal cancer (CRC). The present review assessed recent studies on OSNA vs a histopathological examination and its implications for CRC staging and treatment. A total of 16 studies of OSNA in CRC yielded by a PubMed search were reviewed. Among them, seven studies evaluating the diagnostic performance revealed that OSNA had a high specificity (96.8%), high concordance rate (96.0%), and negative predictive value (98.6%) in a pooled assessment. In addition, four studies examining the utility of OSNA in sentinel LNs (SLNs) and two studies focusing on upstaging in pathologically node‐negative CRC patients were also reviewed. Multicenter prospective studies with a large cohort of CRC patients are warranted to reveal the benefits of OSNA in the future. Although the validity of one‐step nucleic acid amplification (OSNA) assay for detecting lymph node metastasis has been widely reported in patients with other types of malignancies, such as colorectal cancer (CRC), lung cancer, gastric cancer, head and neck squamous cell carcinoma, and thyroid cancer, its clinical benefit has not been established. We herein review studies relevant to OSNA in CRC and discuss perspectives on the future applications of this assay. OSNA was first reported in breast cancer patients and has been shown to be effective for detecting nodal metastases.Although the validity of OSNA assay for detecting LN metastasis has also been widely reported in patients with other types of malignancies, such as CRC, lung cancer, gastric cancer, head and neck squamous cell carcinoma (HNSCC), and thyroid cancer, its clinical benefit has not been established.We herein review studies relevant to OSNA in CRC and discuss perspectives on the future applications of this assay.2A search for medical reports relevant to OSNA in colorectal cancer published before the end of June 2020 was made through PubMed using the keywords of “OSNA” and “colorectal cancer” or “colon cancer” or “rectal cancer.” Reports written in languages other than English were excluded. Review articles were also excluded. The 16 studies obtained by the PubMed search are shown in Table 3At present, the validity of the OSNA assay targeting CK19 mRNA for detecting LN metastasis has been widely reported in patients with various types of tumors, including CRC. Yamamoto et al reported the background for the identification of CK19 mRNA as an optimal marker for the OSNA assay in CRC.4Among the 16 studies listed in Table It should be noted that the methodology of the histopathological evaluation for detection of the LN metastasis differed among studies. For example, a standard pathological examination using HE staining with a single 4‐µm‐thick tissue section was used in some studies,The studies listed in Table 5In CRC, the SLN concept was introduced in 1999 by Joosten et alThe principle of an SLN biopsy is well established in melanoma and breast cancer, where the aim is to avoid unnecessary and potentially morbid lymphadenectomy. Unlike these two malignancies, where lymphadenectomy is a separate procedure, lymphadenectomy in elective CRC surgery is typically performed as part of a single surgical procedure. Therefore, an SLN biopsy to omit unnecessary lymphadenectomy does not seem clinically useful in CRC surgery. However, if an SLN biopsy can readily and reliably determine the LN status, permitting more conservative surgery, then reduced tissue dissection, shortened operative time, and a better bowel function are all desirable outcomes, especially in early‐stage CRC patients.The main advantage of SLN mapping in CRC appears to be the identification of nodes that carry an increased risk of metastasis, as these LNs can then be subjected to detailed scrutiny, including more sections and IHC, thereby optimizing the staging accuracy. While whether or not LN mapping can improve the outcomes for CRC patients is unclear because the influence of the detected micrometastasis on the survival and therapeutic decision is controversial, different studies have shown that ex vivo SLN mapping is an easy and feasible technique for CRC patients. This technique was characterized by a high accuracy of 90%‐100% and NPV of 80%‐100%,Previous studies analyzing CRC metastases in SLNs with the OSNA are listed in Table The average total time needed to complete the OSNA assay is approximately 40 minutes,6Patients with stage II CRC (T3‐4N0M0) form a heterogeneous group with very different prognoses, and some patients with stage II CRC may have a worse prognosis than patients with low‐risk stage III CRC (T1‐3N1).Although the prognostic value of an OSNA analysis in stage II CRC is still unclear compared with that of ctDNA, more recent studies have hypothesized that positive results on an OSNA assay in stage II CRC patients correlate with a poor disease prognosis. Brito et al examined 753 LNs from pathologically node‐negative colon cancer patients (n = 59) by an OSNA assay and showed that 28.8% of the patients had OSNA‐positive LNs.Another study by Itabashi et al evaluated the prognosis of CRC patients with pathologically negative (pStage II) OSNA‐positive LNs.7In Western countries, preoperative chemoradiotherapy (CRT) with total mesorectal excision (TME) is the standard therapy for rectal cancer.8The LN status remains the most significant prognostic factor and an important component of the decision‐making process regarding the adjuvant treatment for CRC cancer patients. In that respect, OSNA can be useful as an accurate, reproducible and automated assay for standardizing the LN metastasis evaluation. In addition, the OSNA may have the potential to detect occult metastasis that cannot be detected by a histopathological examination. Furthermore, Diaz‐Mercedes et al analyzed the budget impact of introducing an OSNA assay in early‐stage CRC patients and suggested that OSNA might have not only an economic benefit but also a clinical benefit in CRC patients, since it enabled more accurate staging, thereby avoiding unnecessary treatment.However, one disadvantage of the OSNA assay is that harvesting LNs requires crude manual dissection of the fresh specimen by a trained pathologist or technician. This step is crucial for any analysis but is not uniform, so missing nodes is unavoidable. Another disadvantage of OSNA assay is that using the whole LN means there is no tissue left for a subsequent examination following complete homogenization, making it impossible to conduct multiple tests of the LN for different biomarkers in the future.The OSNA assay is useful for determining the treatment strategy for CRC patients, as summarized above. In addition, there are several potential advantages worth evaluating in the future. For example, conducting an OSNA assay to detect LN metastasis in rectal cancer patients who have undergone preoperative CRT and surgery might be useful. The presence of residual LN metastasis despite the involutional effect of CRT has been shown to be a major risk factor of local recurrence, distant recurrence, and poor survival.The main advantage of an OSNA assay over a pathological examination is that it provides objective and semi‐quantitative data on the tumor volume in the whole LN rapidly with little to no effort by pathologists and with low inter‐observer variability. As described above, an OSNA assay can help improve the treatment outcome of CRC patients in several respects, such as in the detection of high‐risk stage II disease, the SLN analysis and the prediction of LPLN metastasis. Multicenter prospective studies with a large cohort of CRC patients are warranted to confirm such advantages of an OSNA assay, and technical refinements of the OSNA system are essential before it can be applied as a new standard.Conflicts of interest: Authors declare no conflicts of interest or financial ties to disclosure.Author Contribution: Yukiharu Hiyoshi study conception design, Drafting the article. Takashi Akiyoshi study conception design. Yosuke Fukunaga study conception design.