Peripheral artery disease (PAD) is a common and debilitating condition characterized by the narrowing of the limb arteries, primarily due to atherosclerosis. Non-invasive multi-modality imaging approaches using computed tomography (CT), magnetic resonance imaging (MRI), and nuclear imaging have emerged as valuable tools for assessing PAD atheromatous plaques and vessel walls. This review provides an overview of these different imaging techniques, their advantages, limitations, and recent advancements. In addition, this review highlights the importance of molecular markers, including those related to inflammation, endothelial dysfunction, and oxidative stress, in PAD pathophysiology. The potential of integrating molecular and imaging markers for an improved understanding of PAD is also discussed. Despite the promise of this integrative approach, there remain several challenges, including technical limitations in imaging modalities and the need for novel molecular marker discovery and validation. Addressing these challenges and embracing future directions in the field will be essential for maximizing the potential of molecular and imaging markers for improving PAD patient outcomes. Peripheral artery disease (PAD) is a common and debilitating condition characterized by the narrowing and obstruction of the antegrade flow of major systemic arteries other than those of the cerebral and coronary circulations, primarily due to the buildup of atherosclerotic plaques. PAD affects over 230 million people worldwide and is associated with significant morbidity, mortality, and reduced quality of life .Several factors have been identified as major contributors to the development and progression of PAD. These include non-modifiable risk factors such as advanced age and genetic predisposition and modifiable ones such as smoking, diabetes, hypercholesterolemia, and hypertension. Lifestyle elements such as poor diet, physical inactivity, and obesity also play considerable roles ,3.Central to PAD’s pathogenesis is atherosclerosis, which is initiated when low-density lipoprotein (LDL) cholesterol accumulates within the artery walls, triggering an inflammatory response. This response leads to the recruitment of immune cells, primarily monocytes, that transform into macrophages, ingest the accumulated lipids, and become foam cells. Over time, the accumulation of these foam cells forms a lipid core within the arterial wall, which constitutes the atherosclerotic plaque .Endothelial dysfunction is another key factor that promotes atherosclerosis in PAD. A healthy endothelium maintains vascular homeostasis by regulating vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and inflammation. However, in conditions such as hypertension, hypercholesterolemia, and diabetes, the endothelium becomes dysfunctional, promoting the adhesion and infiltration of inflammatory cells and the formation of atherosclerotic plaques .As plaques grow and arterial stenosis worsens, blood flow to the affected limb is significantly reduced, leading to the clinical manifestation of PAD. Symptoms can range from asymptomatic disease to intermittent claudication—characterized by muscle pain or cramping during physical activity—to critical limb ischemia, where the blood flow is so poor that it leads to gangrene, necessitating limb amputation. Moreover, these plaques can become unstable and rupture, leading to acute limb ischemia, a severe and painful condition that requires immediate attention ,5.Early detection and accurate characterization of PAD are crucial for effective risk stratification, appropriate therapeutic intervention, and monitoring treatment response. In the landscape of diagnostic tools for peripheral arterial disease (PAD), traditional methods such as the ankle–brachial index (ABI) and pulse wave velocity (PWV) measurements have long served as reliable indicators. ABI, a straightforward, non-invasive tool, offers a quantifiable measure of the presence and severity of PAD. It accomplishes this by comparing the blood pressure in the ankle to the blood pressure in the arm, thus reflecting the adequacy of the blood flow ,6. HowevOn the other hand, PWV is a measurement of arterial stiffness, which has been recognized as a potential predictor of PAD. PWV measurements are obtained by capturing the velocity of the pressure waveform between two points along the arterial tree ,8. WhileWith the advent of advanced imaging technologies, the ability to non-invasively visualize and quantify PAD has significantly improved. Non-invasive medical imaging modalities, such as computed tomography (CT), magnetic resonance imaging (MRI), and nuclear imaging, such as single-photon emission computerized tomography (SPECT) and positron emission tomography (PET), have emerged as valuable tools for imaging PAD atheromatous plaques and vessel walls. These modalities provide detailed information on plaque burden, morphology, and composition and insights into vascular function and blood flow. Despite their higher cost and less widespread availability, these newer modalities hold the potential to revolutionize PAD diagnostics and prognostics. Furthermore, integrating molecular markers with imaging markers from these modalities can enhance our understanding of PAD pathophysiology and improve patient outcomes.In this review, we will examine the applications of CT, MRI, and nuclear imaging in assessing PAD, focusing on the imaging markers derived from each modality and their potential correlations with molecular markers. We will also discuss the advantages and limitations of each imaging modality, the role of molecular markers in PAD pathophysiology, and the potential benefits of integrating molecular and imaging markers in the comprehensive assessment of PAD.An English-language literature search was conducted on PubMed, limited to articles published since 2000. The search was carried out in two stages: initially on 13 January 2023 and subsequently updated on 1 May 2023. A combination of title, abstract keywords , and Medical Subject Headings  were used to identify relevant studies that either evaluated or aimed to evaluate PAD using non-invasive medical imaging modalities or correlated imaging biomarkers with molecular markers of PAD.In addition, reference lists from selected articles were examined to identify further applicable studies, and current practice guidelines were reviewed. Articles were selected for inclusion based on the quality of the research and its relevance to the review’s theme. For instance, studies that did not utilize non-invasive medical imaging modalities were excluded.The search strategy yielded a total of 294 articles. From these, 14 pertaining to CT, 29 to MRI, 1 to SPECT, 7 to PET, and 6 studies discussing the correlation between imaging and molecular markers were selected for inclusion in this review.This section briefly overviews the primary non-invasive medical imaging modalities used for imaging PAD plaques and vessel walls: CT, MRI, and nuclear imaging. For each imaging modality, we discuss the basic principles, advantages, and limitations .CT imaging uses fan-beam X-rays and computer processing to generate cross-sectional images of the body’s internal structures . It offeMRI relies on nuclear proton magnetic resonance and the interaction of magnetic fields, radiofrequency pulses, and gradients to create detailed images of the body’s internal structures . MRI offNuclear imaging involves injecting radiotracers to visualize the distribution and biochemical function of specific molecules or cells within the body. The two main types of nuclear imaging used in PAD assessment are positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Nuclear imaging can visualize molecular and cellular processes in vivo by providing insights into plaque metabolism, inflammation, and neovascularization depending on the injected radiotracer ,19. HoweIn summary, CT, MRI, and nuclear imaging each offer unique advantages and limitations for PAD imaging. The choice of the most appropriate modality depends on the clinical context and the specific objectives of the assessment, such as plaque detection, characterization, or evaluation of treatment response. In the following sections, we will delve deeper into the applications and imaging markers associated with each modality and their potential integration with molecular markers for a comprehensive assessment of PAD.In this section, we discuss the applications of CT in assessing PAD. With technological advances and accessibility, CTA has become a popular choice for PAD evaluation. CTA utilizes the emission and detection of X-rays as the patient passes through a gantry that rotates the X-rays in a 360° arc to generate three-dimensional data . An intrCTA is extensively employed to evaluate PAD, as it facilitates detailed visualization of the arterial lumen , detectiDespite its limited diagnostic ability in the below-knee region, CTA has several advantages over DSA. Firstly, unlike catheter-based DSA, CTA is non-invasive and reduces catheter-associated patient risks and operator dependency. Additionally, although both DSA and CTA involve ionizing radiation, the radiation dose exposure of CTA is significantly lower than that of DSA . FurtherIn addition to evaluating luminal narrowing, stenosis, and the extent of lesions, CT also provides information about vascular and plaque calcification, which is crucial for risk stratification in PAD . Patel eCT studies have also revealed that diabetes, a common underlying condition of PAD, is associated with increased lower limb vascular calcification ,35. In 2Although CTA-derived vascular and plaque calcification aids in PAD risk stratification, the presence of vessel wall calcification often hinders accurate interpretation of peripheral artery CTA examinations. Streak and blooming artifacts caused by vessel wall calcification can lead to overestimating the vessel stenosis . It has 3 µm3) in both carotid and lower limb arteries. However, they found that weight-based extra-coronary calcium scores (ECCS) of both carotid and lower limb arteries had only weak positive correlations with the distribution of calcified particles  and microcalcifications   tetrofosmin [99mTc] diphosphonate, [99mTc] anti-granulocyte antibodies, and indium-111 (111In)-labeled leukocytes sestamibi and [99mTc]tetrofosmin are the most commonly used radiotracers for evaluating lower extremity skeletal muscle perfusion CO2 and 15O2 inhalation revealed asymmetrical patterns of calf muscle blood flow in PAD patients during ankle flexion. Subsequent studies using [15O]H2O PET imaging successfully quantified absolute calf muscle perfusion at rest, during exercise, and after infusion of prostaglandin E1 in healthy subjects and PAD patients. PET-derived measures of muscle blood flow have been shown to correlate closely with traditional blood flow assessment techniques. Ongoing developments with modern PET imaging systems are expected to bring additional opportunities for non-invasive imaging of PAD and improve the quantitative assessment of lower extremity perfusion in the setting of limb ischemia ..18F-FDG 2 is another inflammatory protein produced by inflammatory cells to hydrolyze oxidized phospholipids on the surface of LDL particles. Previous studies have found that increased Lp-PLA2 mass and activity predict an increased risk for PAD incidence and low ABI . S. Smds, aApart from the aforementioned categories, other biomarkers have been linked to PAD. Angiogenesis factors, such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α, promote capillary network growth in response to local hypoxia in the ischemic limb of the PAD . In 2015Considering the role of vascular calcification in PAD evaluation, vascular calcification markers have also garnered attention. Matrix Gla-Protein (MGP) is a vitamin-K-dependent vascular calcification inhibitor, and inactive MGP is associated with arterial stiffness and increased risk for PAD ,163,164.In summary, various molecular markers, including those related to inflammation, endothelial dysfunction, oxidative stress, and other processes, have been implicated in PAD pathophysiology. These markers hold promise as diagnostic and prognostic indicators. Several studies have demonstrated correlations between these molecular markers and imaging markers, which further enhanced our understanding of PAD and potentially contributed to developing personalized treatment strategies.This section discusses the challenges of integrating molecular and imaging markers in assessing PAD and explores potential future directions in the field. These future directions include advancements in imaging technologies and the discovery of novel molecular markers that can enhance our understanding of PAD pathophysiology.One of the primary challenges in PAD imaging is addressing the limitations of current imaging modalities, such as spatial resolution, sensitivity, and specificity. Accurate characterization of PAD plaques and vessel walls is crucial for a comprehensive understanding of the disease. Additionally, standardizing imaging protocols and quantifying imaging markers for comparison across studies remain significant challenges. For example, MRA has been accused of producing different results across different institutes, limiting its application in clinics and multi-center research ,59. Over18F-FDG uptake did not correlate with plaque macrophage content and activity, suggesting a unique pathophysiology of PAD and a need for specific markers [In terms of molecular markers, there is a need to identify and validate novel markers that are specific and sensitive to PAD pathophysiology. Unlike carotid and coronary plaques, previous studies found that femoral artery plaque  markers ,146. HowEstablishing a robust correlation between molecular and imaging markers in PAD is essential for maximizing their diagnostic and prognostic potential. Integrating molecular and imaging data can provide a more comprehensive perspective on the underlying mechanisms of the disease and the factors that contribute to its progression. However, further research is needed to explore the benefits and limitations of this integrative approach, as well as to determine the most effective methods for combining these data sources.In summary, addressing the challenges associated with integrating molecular and imaging markers in the assessment of PAD is vital for enhancing our understanding of the disease’s pathophysiology. Embracing future directions in this field, such as advancements in imaging technologies and the discovery of novel molecular markers, will be essential for maximizing the potential of these markers in improving PAD patient outcomes. By overcoming these challenges, researchers and clinicians can work towards developing more effective diagnostic and prognostic tools for PAD.Significance and Impact: PAD poses a substantial clinical challenge, the prognosis of which depends heavily on effective diagnostic and monitoring methodologies. Current investigations and the available literature demonstrate the importance of non-invasive imaging techniques and molecular markers in evaluating the status and progression of the disease.Plaque Composition Analysis: Comprehensive understanding of the composition of atheromatous plaques is crucial in PAD management. Techniques such as high-resolution MRI and CT angiography, complemented with the analysis of biomarkers such as inflammatory markers and oxidative stress markers, yield important insights into the nature and characteristics of plaques.Extent of Atherosclerotic Plaques: Imaging techniques such as CT angiography and MRI provide effective means to quantify the extent of atherosclerotic plaques in PAD patients. Concurrent assessment of molecular markers, especially those indicative of endothelial dysfunction, enriches our understanding by offering a molecular perspective of the ongoing pathological process.Activity of Atherosclerotic Process: The ability to monitor the activity of the atherosclerotic process is crucial in tracking disease progression and evaluating the effectiveness of therapeutic interventions. The combined application of non-invasive imaging techniques and the analysis of inflammation and oxidative stress markers can provide a reliable indication of the disease activity status.Significance of Stenosis: The severity of stenosis in PAD is a key determinant of prognosis and therapeutic planning. Precise stenosis evaluation can be achieved with modern imaging modalities such as CT and MRI. Combined with biomarker analysis, these tools can provide clinicians with a robust basis for predicting outcomes and formulating individualized treatment strategies.Future Perspectives: The future holds promising advancements in non-invasive imaging and molecular marker research, with potential benefits for PAD management. Emerging techniques such as PET and SPECT, along with novel molecular markers, are set to enhance our understanding of the disease and its management.Integrating non-invasive imaging techniques with molecular markers is vital for comprehensively understanding and managing PAD. It is our hope that the ongoing advancements in this field will continue to improve our understanding of the pathophysiological processes underlying PAD, ultimately resulting in improved patient care.


HB(G) of a graph G are computed.The novel applications in chemistry include the mathematical models of molecular structure of the compounds which has numerous findings in this area that refers to mathematical chemistry. Topological descriptors play a major role in QSAR/QSPR studies that analyses the biological and physicochemical properties of the compounds. In the recent times, a new type of topological descriptors are proposed, called K-Banhatti indices. In this study the chemical applicability of K-Banhatti indices are examined for benzenoid hydrocarbons (derivatives of benzene). These indices have shown remarkable results through the study of statistical analysis. Subsequently, triazine-based covalent organic frameworks (CoF’s) are studied for which  There are varied applications in chemistry and these quantities are derived from their molecular structure. The chemical information obtained from the molecular descriptors vary for different algorithms proposed according to its respective definitions. Using the algorithm of a particular molecular descriptor, a detailed description of a molecule can be obtained. The selection of a molecular descriptor depends on the problem on which it is applied. The key technique is in encoding the information obtained from molecular descriptors using the structure of the molecule2.The modelling and forecasting of physicochemical and biological properties of molecules is aimed in the studies of Quantitative structure–property relationships (QSPR). Statistical and mathematical tools are used to extract every possible information about a compound using the help of chemometrics. The variation of physicochemical property of a molecule with respect to the topological index can be described using chemometrics through QSPR. This can replace expensive biological tests conducted in a laboratory, especially when the experiments involve hazardous and toxic materials or unstable compounds. An optimum relationship in predicting the properties of compounds is the basic strategy of QSPR. The performance of this study depends on the description of the molecular structure and their parameters6. The molecular descriptors are used to extract most of the information of a compound using simple and quick computations.To encode the information of a chemical structure, several topological indices were developed. These indices draw attention as they play a significant role in the contributions of QSPR studies12.Topological descriptors have a crucial role in QSAR/QSPR studies that analyses the biological and significant properties of the compounds. The mathematical chemistry is a combination of modelling of chemical compound to derive pivotal data through the various tools viz., topological indices, QSAR/QSPR studies and various polynomials14, Kulli proposed various novel degree-based TIs such as the first K-Banhatti index HB(G)) of a graph G are defined asu, and an edge e are incident in the graph G and numerous studies have been carried out on these indices so far19. But it is noticed that the chemical applicability of these indices have not been studied yet. Furthermore, the flowchart of the metodology defined above is shown in the Fig. 23 . Subsequently, triazine-based covalent organic frameworks (TriCF) are studied for which the first K-Banhatti index, the second K-Banhatti index, first K-hyper Banhatti index, the second K-hyper Banhatti index, the modified first K-Banhatti index, the modified second K-Banhatti index, and the harmonic K-Banhatti index of a graph G are computed.Recently in Refs.V and E are known as the order, and the size of the graph G respectively. The cardinality of edges incident to a vertex e denoted by, u and an edge e are adjacent in the graph G26.In this paper, consider the simple and connected graph 28 for the considered indices using Tables R with the above four properties show high positive correlation  are highly correlated with the The linear regression models for the first K-Banhatti index(The linear regression models for the second K-Banhatti index(The linear regression models for the first K-hyper Banhatti index(The linear regression models for the second K-hyper Banhatti index(The linear regression models for the modified first K-Banhatti index(The linear regression models for the modified second K-Banhatti index(HB) The linear regression models for the harmonic K-Banhatti index(This section concentrates on framing the linear regression model for the properties such as boiling point (BP), enthalpy (E), mentclass2pt{minimThe chemical systems that possess discrete number of molecules refers to supra-molecular chemistry. The spatial arrangement of the molecules is responsible for the strength of the forces between them may be weak or strong. These forces may be due to intermolecular, hydrogen bonding, electrostatic charge, and covalent bonding. The feeble and reversible non-covalent interactions between the molecules are examined by the supramolecular chemistry while traditional chemistry examines covalent bonds. Various functions of supramolecular chemistry comprise molecular recognition, protein folding, interlocked molecular architectures, dynamic covalent chemistry, and other phenomena. Because of its interdisciplinary nature, it attracts physicists, biochemists, biologists, environmental scientists, apart from chemists.33.This work pinpoints on the supramolecular structure called triazine-based covalent organic frameworks Fig. . To undeBiologically significant organic molecules have a new dimension as triazines act as the building blocks used in its design. Triazines and its derivatives have varied applications in antifungal, anticancer, antiviral, cardiotonic, anti-HIV, analgesic, etc., with fine tuned electronic properties. The goal of scientific researchers is to apply their theoretical research in industrial applications so that it is useful for humankind. The objective is to make the products scalable and satisfy excellent properties obtained from the experiments at a reasonable cost and long-term stability.37. CoF’s may be classified into boron-containing, triazine-based, imide-linked and imine-based due to swift increasing requirements in various fields. We focus on the second category of CoF’s, i.e., triazine-based in this study. In 2008, Thomas et al.38, prepared triazine based CoF, by cyclotrimerization of nitrile building units at 400 39.Covalent organic frameworks (CoF’s) have attracted various researchers across the globe because of its excellent properties such as adsorption, chemo-sensing, energy storage and production. As the CoFs and their applications are found in industries, many research achievements have come to light recently40. It was noted that 2-dimensional CoF’s acts as catalyst in different reactions that include nitrophenol reduction, water oxidation, in reducing CO42.The distinguishing physical and chemical features of CoF’s have led to the plethora of applications in the industries. In 2011, Ding et al. reported first set of CoF’s that are useful in the field of catalysis44 theoretically examined triazine-based covalent-organic frameworks (CoF’s) using vertex and edge partition for degree-based and neighborhood degree-based topological indices. Additionally, the degree-based and neighborhood degree-based entropy measures for the results are given. The graph theoretical approach is used to compare the outcomes with obtained results. In this section, based on the previous work of Tony Augustine et al. various K-Banhatti topological indices are computed for triazine-based covalent-organic frameworks (TriCF).Augustine et al.G be the the molecular graph of linear chain TriCF. Then,HB(G) of a graph G areLet 44)From the Fig. G be the the molecular graph Parellelogram TriCF. Then HB(G) of a graph G areLet 44)From the Fig. G be the the molecular graph Hexagonal TriCF. Then HB(G) of a graph G areLet 44)From the Fig. Figures Figures Table  The molecules are modelled, and their physicochemical and biological properties are predicted using the Quantitative structure-property relationships (QSPR) studies. Topological index is a significant tool used by QSPR studies in encoding the information of a molecule. There are a bunch of topological indices which are of significant importance in the properties of the compounds based on its algorithm defined.HB(G) for benzenoid hydrocarbons of a graph G are examined and it is observed that the considered indices (molecular descriptors) showed good predictive potential. Benzenoid hydrocarbons have numerous applications because of its unique physical and chemical properties. Some of them include paint thinners, laminates, cement, in medicine for curing bacterial infections, mosquito repellents, cosmetics, toothpaste, detergents, and a dyeing agent.In this article, chemical applicability of Also the above said indices are computed for triazine-based covalent organic frameworks (CoF’s). Triazine has wide applications in industries, where one of the famous forms being melamine. It is used in kitchen appliances and carpentry. Another form of triazine is cyanuric chloride that are used in reactive dyes and herbicides. It has several applications in oil, petroleum and gas processing industries. They are used to remove harmful hydrogen sulphide gas and other species from fluid streams in infrastructure. As the chemical compound, triazine has many applications especially in industries, the work can be extended for other indices using graph operators and see the variation. Also, it has applications in medical field, attracting the pharmacists and chemists in the usage of drug design and delivery.


Emerging research has reported that circular RNAs (circRNAs) play important roles in cardiac cell death after myocardial ischemia and reperfusion (I/R). Ferroptosis, a new form of cell death discovered in recent years, has been proven to participate in the regulation of myocardial I/R. This study used circRNA sequencing to explore the key circRNA in the regulation of cardiac ferroptosis after I/R and study the mechanisms of potential circRNA function.We performed circRNA sequencing to explore circRNAs differentially expressed after myocardial I/R. We used quantitative polymerase chain reactions to determine the circRNA expression in different tissues and detect the circRNA subcellular localization in the cardiomyocyte. Gain- and loss-of-function experiments were aimed to examine the function of circRNAs in cardiomyocyte ferroptosis and cardiac tissue damage after myocardial I/R. RNA pull-down was applied to explore proteins interacting with circRNA.Fth1), a ferroptosis suppressor, which resulted in the inhibition of cardiomyocyte ferroptosis.Here, we identified a ferroptosis-associated circRNA (FEACR) that has an underlying regulatory role in cardiomyocyte ferroptosis. FEACR overexpression suppressed I/R-induced myocardial infarction and ameliorated cardiac function. FEACR inhibition induces ferroptosis in cardiomyocytes and FEACR overexpression inhibits hypoxia and reoxygenation-induced ferroptosis. Mechanistically, FEACR directly bound to nicotinamide phosphoribosyltransferase (NAMPT) and enhanced the protein stability of NAMPT, which increased NAMPT-dependent Sirtuin1 (Sirt1) expression, which promoted the transcriptional activity of forkhead box protein O1 (FOXO1) by reducing FOXO1 acetylation levels. FOXO1 further upregulated the transcription of ferritin heavy chain 1 (Our finding reveals that the circRNA FEACR-mediated NAMPT-Sirt1-FOXO1-FTH1 signaling axis participates in the regulation of cardiomyocyte ferroptosis and protects the heart function against I/R injury. Thus, FEACR and its downstream factors could be novel targets for alleviating ferroptosis-related myocardial injury in ischemic heart diseases.The online version contains supplementary material available at 10.1186/s12929-023-00927-1. Heart disease is a major killer that is seriously harmful to health, according to a report from the American Heart Association . AlthougCircular RNAs (circRNAs), which are a type of noncoding RNA, were first discovered in plant viruses in 1976 . CircRNAFth1) transcription. Our findings reveal a novel regulatory role of circRNAs in ferroptosis and identify a new molecular mechanism of circRNAs in the treatment of heart diseases.Our present study revealed that the circRNA FEACR (ferroptosis-related circRNA) is involved in the regulation of cardiomyocyte ferroptosis and improves myocardial I/R injury. We found that FEACR directly interacts with nicotinamide phosphoribosyl transferase (NAMPT) and maintains its stability, thus affecting NAD-dependent deacetylase sirtuin1 (Sirt1)-mediated forkhead box protein O1 (FOXO1) deacetylation to regulate ferritin heavy chain 1  supplemented with 5% fetal bovine serum was used to resuspend the pellet. The cell suspension was filtered with a 100 mesh filter in a 10 cm dish. The cells were adherent in an incubator for 1.5 h and suspending cells were primary cardiomyocytes. The cardiomyocytes were cultured in a 5% DMEM/F12 medium with 0.1 mM bromodeoxyuridine in an atmosphere of 5% CO2 at 37 °C.Newborn mice aged 1–3 days were used to isolate primary cardiomyocytes. First, we executed the mice, harvested hearts, and collected them in a 6-well plate. All the isolated hearts were washed with phosphate buffer saline (PBS) three times and cut into tissue pieces of 1mm2 and 95% N2) at 37 °C for 18 h, Subsequently, the cells were cultured in 5% medium and placed in an incubator (95% air and 5% CO2) at 37 °C for 6 h to establish in vitro H/R model.H/R was performed in cardiomyocytes as previously describe , 10. In We purchased the adult male C57BL/6J mice (8–10 weeks old) from Pengyue Experimental Animal Breeding Co., LTD . The mice used for the experiment were of the same age and body weight and were assigned randomly to each group. All animal researches conform to the Directive 2010/63/EU of the European Parliament and have been approved by the Biomedical Research Ethics Committee of Qingdao University (License No: 20200610C576620221208124).10 moi) by tail vein injection. For knockdown of FEACR, NAMPT, and FTH1, the mice were treated with adv-shFEACR, adv-shNAMPT, and adv-shFTH1 (2 × 1010 moi) by tail vein injection. Three days after injection, mice were treated with I/R, and heart samples were collected to conduct molecular and morphological studies. We conducted the experiments in accordance with the guidelines of Qingdao University’s Animal Care Committee.The ischemia and reperfusion (I/R) model is conducted by ligation of the left anterior descending coronary artery (LAD) for 45 min and reperfusion for 3 h, as previously described . In the RIPA buffer containing 100 μM PMSF (Solarbio) is used to lyse cardiomyocytes and cardiac tissues. Cardiomyocytes or cardiac tissues were suspended in RIPA buffer and were lysed on ice for 20 min. The lysate was centrifuged at 4 °C, 13,000 rpm for 20 min, and the concentration of total protein was measured by BCA kit (Vazyme). SDS-PAGE electrophoresis was used to separate cell lysates, which were then transferred onto PVDF membranes. The primary antibody was incubated with the membrane at 4 °C overnight, the blot was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature followed by development with ECL Western blot substrate. We use primary antibodies in the following concentrations: GPX4  1:3000, SLC7A11  1:3000, FTH1  1:2000, NAMPT  1:3000, Sirt1  1:2000, FOXO1  1:1000, Ac-FOXO  1:1000, RIPK1 1:2000, RIPK3  1:2000, MLKL  1:2000, BAX  1:1000 and GAPDH  1:5000, overnight at 4 °C.2, 1 mM DTT, 1× protease inhibitors) and digested for 30 min. The lysates were centrifuged at 12,000 rpm, 4 °C for 20 min, and aliquoted 50 μL lysates were used as input. The remaining lysates were incubated respectively with biotin-FEACR and biotin-NC and formed a mixture overnight. A solution of streptavidin-magnetic beads (BioMag) was then incubated with mixture at 4 °C for 3 h and washed five times with lysis buffer. Then the beads were boiled with 2× SDS loading buffer (SparkJade) for 5 min at 95 °C. The PAGE gel was stained with coomassie bright blue until the band is clear and extracted gel lines underwent LC–MS/MS analysis at a commercial laboratory. The gel band was digested by trypsin and HPLC liquid system Easy nLC was used to separate each sample at a nanoliter flow rate. After chromatographic separation, mass spectrometry was applied to detect the samples by using a Q-Exactive mass spectrometer. The original data of mass spectrometry analysis were RAW files, and MaxQuant software (version No. 1.5.3.17) was used for database identification and quantitative analysis (Shanghai Applied Protein Technology). The raw MS data was uploaded to the iProX database, and the subproject ID is IPX0005237002.A FEACR labeled with 5′-biotin and negative control (NC) were obtained from GenePharma, the sequence as follows: Biotin-FEACR: 5′biotin-AAUGCCUGCCAUGCUGAGUAAUGGG; Scramble Biotin-FEACR: 5′biotin-UAAUGCACGAUGUGGUCGUAACGGC. The cardiomyocytes were washed with PBS several times and then collected in a 1.5 mL EP tube. The cell pellet was resuspended in lysis buffer  for multiple comparisons. Statistical analyses were performed using GraphPad Prism software , and P-value < 0.05 were considered significant.P < 0.05) and confirmed their expression levels by RT‒qPCR. The results showed that the expression of the top ten upregulated RNAs was not significantly altered by I/R or H/R  and is derived from the back-splicing of linear FBXW4 mRNA exons 2–5. We experimentally verified the ring structure of FEACR in mouse hearts using divergent primers and RT‒PCR  mice and confirmed the delivery efficiency Fig. a. Evans roptosis . FEACR oPtgs2 expression , which inhibits ferroptosis in cardiomyocytes by targeting the NAMPT-mediated Sirt1-FOXO1 pathway. Enforced expression of FEACR attenuates ferroptosis of cardiomyocytes caused by H/R and improves I/R-induced myocardial infarction and ameliorates cardiac function. Mechanistically, FEACR directly binds to NAMPT and enhances the protein stability of NAMPT. This leads to the increase in NAMPT-dependent Sirt1, which promotes the transcriptional activity of FOXO1 by reducing its acetylation levels. FOXO1 further upregulates the transcription of Additional file 1: Supplemental figures and methods.Additional file 2: Table S1. List of differentially circRNA in I/R-operated mice heart compared to sham heart.Additional file 3: Table S2. List of biotinylated FEACR versus NC pull-down proteins identified by MS.Additional file 4: Table S3. The primer pairs used for quantitative real-time PCR analysis of gene expression.


The development of nanotechnology has provided an opportunity to integrate a wide range of phenomena and disciplines from the atomic scale, the molecular scale, and the nanoscale into materials. Nanoarchitectonics as a post-nanotechnology concept is a methodology for developing functional material systems using units such as atoms, molecules, and nanomaterials. Especially, molecular nanoarchitectonics has been strongly promoted recently by incorporating nanotechnological methods into organic synthesis. Examples of research that have attracted attention include the direct observation of organic synthesis processes at the molecular level with high resolution, and the control of organic syntheses with probe microscope tips. These can also be considered as starting points for nanoarchitectonics. In this review, these examples of molecular nanoarchitectonics are introduced, and future prospects of nanoarchitectonics are discussed. The fusion of basic science and the application of practical functional materials will complete materials chemistry for everything. Nanotechnology is a game changer that has innovated the course of scientific research. Nanotechnology innovations have unlocked mysteries at the nanoscale –3. These2 complexes on carbon nanohorns has been reported . T. Tb:4,5-60 fullerene molecules  surface using a combination of low-temperature scanning tunneling microscopy/spectroscopy and density functional theory calculations . I. I119]. 4Si2) backbones (x film. Bromo-substituted polycyclic hydrocarbon precursors (triphenylene or pyrene) were then deposited on this surface and annealed to form a C4Si2 bridging network. In the linear structures obtained with pyrene precursors, the C4Si2 rings were converted to C4Si pentagonal siloles by further heat treatment. These results demonstrate that coupling nanoarchitectonics on C–Si surfaces is possible by depositing Si atoms and, subsequently, polycyclic hydrocarbons on Au(111). It is expected that various low-dimensional nanostructures will be synthesized by this on-surface synthetic nanoarchitectonics.In organic molecules and materials, the electronic structure and physical properties can be modified by replacing carbon with silicon. For example, silicon-substituted graphene-based materials exhibit exotic properties. However, it is difficult to synthesize silicon-substituted conjugated organic materials with atomic precision using conventional organic synthesis methods. Foster, Kawai, and co-workers have successfully synthesized one-dimensional and two-dimensional covalent organic frameworks on the surface of 1,4-disilabenzene (Cackbones  120]. F. F4Si2) The bottom-up synthesis of graphene nanoribbons on surfaces has attracted much attention due to their high electronic, optical, and magnetic properties. Sakaguchi and co-workers have synthesized cove-shaped two-dimensional graphene nanoribbon networks by interconnecting one-dimensional self-assembled graphene nanoribbons on a Au(111) surface . The strIn addition to bandgap engineering of porous graphene nanoribbons via their width and edge arrangement, periodic nanostructures provide a means to control the electronic properties of graphene nanoribbons. Ma, Tan, Wang, and co-workers have synthesized 5,8-dibromopicene on Au(111) surfaces via trans- and cis-coupling to synthesize 8-carbon armchair graphene nanoribbons and nanographene C66 with periodic vacancies on the surface  124]. D. D124]. 3)-substituted 5,15-bis(10-bromoanthracen-9-yl)-10,20-bis(trifluoromethyl)porphyrin precursors , corresponding to Cu and Co porphyrin units, respectively. Next, the authors went one step further and deposited Pd porphyrin molecules on Au(111), where diblock co-oligomers were already present, and annealed them again. It was confirmed that triblock co-oligomers were formed as expected, albeit in relatively small yields. Thus, multiblock oligomers were successfully synthesized by sequential coupling of CF3-substituted copper, cobalt, and palladium porphyrins on the surface. The assembly of different porphyrin oligomers combines multiple functional properties such as gas adsorption and magnetism in a one-dimensional structure. This approach could also be a promising strategy to construct designer nanoscale carbon materials by sequentially linking multiple monomers on a surface. It is expected to contribute to future carbon-based nanoelectronics.Molecular nanoarchitectonics by on-surface synthesis is a powerful tool for structures other than nanographene. Various functional units can be freely combined to obtain new nanostructures. However, it is generally difficult to link units together in a controlled manner, as reactants are often activated simultaneously at a given temperature. Kawai, Ishikawa, Saito, and coworkers have developed a method to synthesize a multiblock copolymer of a porphyrin metal complex on a surface using trifluoromethyl (CFecursors  125]. T. T3)-subJunctions between molecular block units have also been reported with graphene nanoribbons. Nanoarchitectonics of graphene nanoribbon heterojunctions has the potential to be a major technological breakthrough because of the rational design and by virtue of the extraordinary structural and electronic properties of such heterojunctions. However, graphene nanoribbon heterojunction structures made by bottom-up synthesis are usually difficult to incorporate into functional nanodevices because of the random arrangement of the heterojunctions. Bronner, Fischer, Crommie, and co-workers have developed a single hierarchical fabrication strategy via on-surface synthesis of graphene nanoribbons with a single heterojunction interface  126]. T. T126]. Nanocarbons with a defined two-dimensional extent are also attractive targets for on-surface synthetic nanoarchitectonics. Coronoids are compounds possessing both polycyclic aromatic macrocycles and defined cavities. Because of their unique electronic structures, they receive much attention. The electronic properties of these nanoporous graphene-like compounds are modified through changing size and topology of inner and outer rims in their structures. Especially, extended hexagonal coronoids with zigzag outer edges still pose synthetic challenges. Sun, Yan, Yu, and co-workers have successfully synthesized a C144 hexagon with a zigzag outer edge by hierarchical Ullmann coupling and cyclodehydrogenation on a Au(111) surface  127]. T. T127]. Surface covalent organic frameworks are atomically thick sheets of covalently bonded organic building blocks. Expecting unique properties such as low dimensionality, well-defined in-plane structure, and tunable functionality, Wang, Wan, and co-workers reported a method for the nanoarchitectonics of highly ordered surface covalent organic frameworks (COFs) via a solid–vapor interfacial reaction . In thisAlthough the abovementioned examples describe only a part of the past accomplishments, connecting molecules, building up molecules, and organizing molecules can now be accompanied by directly observing the structures. This is due to significant development in nanotechnology that can alter basic thinking about traditional synthetic organic chemistry. Nanotechnological tools can also be used to induce site-specific organic synthesis and alter selectivity, as seen in the example of local probe chemistry. Indeed, it is now possible to pick up specific substrate molecules and make them react at desired molecular sites. This could be the starting point for assembling functional materials from the molecular level. Hence, molecular nanoarchitectonics is a fusion of advanced nanotechnology and conventional organic chemistry.In this review, we have presented several examples of what we consider to be molecular nanoarchitectonics, with typical examples of research in local probe chemistry and on-surface synthesis. As shown in these examples, nanotechnology has made great contributions to organic chemistry, and it is now possible to observe reaction processes at molecular resolution. In addition, organic synthesis can be freely controlled by manipulation at the molecular level using probe tips. These results show that it is possible to freely synthesize substances at the molecular level. These studies also show that local physical properties can be analyzed by measurements with very high resolution. Thus, it is possible to observe organic chemical reactions at the atomic and molecular level and to understand the properties of molecules at that level.The current mainstream of functional material fabrication is a combination of basic technologies such as organic synthesis, self-assembly, or structure fabrication methods such as the Langmuir–Blodgett (LB) method –131 or lThis approach to molecular nanoarchitectonics based on high-resolution observation is a field rooted in basic science. The fusion of this basic science field with the field of pursuing the application of practical functional materials will complete materials chemistry for everything. The choice of materials is diverse and unlimited. In order to position nanoarchitectonics as a method for everything and as a completed form of general materials science, it is necessary to consider a wide range of applications and many combinations and possibilities. In the past, it has been difficult to apply the method to a seemingly infinite number of substances. However, the development of computational methods such as artificial intelligence will make this possible in current science –144. In


The emergence and spread of antimicrobial resistance (AMR) in bacteria are major public health concerns, posing significant global challenges for sustainable food systems . The useCurrently, there is a need for a scientific multidisciplinary and global perspective on the complex challenge of AMR in food systems. Therefore, in March 2022, a special Research Topic, previously proposed to Frontiers, was published titled “Antimicrobial Resistant Pathogens in Sustainable Food Systems: How to Prevent, Detect, or Control Contamination?”. It included a group of immensely committed handling editors and reviewers to supervise the publication of an article collection. This collection of six articles covers a broad range of topics, from the molecular mechanisms of AMR to the development of novel detection methods, risk assessment, and intervention strategies, to prevent and control contamination.Zhao et al. reviewed the main sources of microbial volatile organic compounds (VOCs) and their potential use as an alternative to synthetic fungicides for post-harvest treatment and control of fruit and vegetable diseases. Furthermore, they discussed recent advancements in understanding the mechanisms of antifungal VOCs and the applications of VOCs produced by antagonistic microorganisms .Eiamsam-Ang et al. presented novel information on the genetic sequencing of Salmonella isolates of rare serotypes obtained from farms, slaughterhouses, and retail markets in Thailand between 2011 and 2014. The authors assessed the genetic diversity and relatedness of these isolates. They also characterized their antimicrobial resistance and virulence genes, highlighting the need for appropriate surveillance of food-animal products to reduce public exposure to highly pathogenic, multi-drug resistant Salmonella .The study by Lambraki et al. investigated the factors influencing AMR in Southeast Asia's food system and proposed intervention measures by integrating local expert perspectives. Their results aim to inform policy improvement and managing decisions .Research presented by Mwansa et al. conducted an original cross-sectional study in Zambia, to determine antibiotic resistance patterns of extended-spectrum β-lactamase-producing E. coli isolated from stool samples of workers who work at poultry farms and to raise awareness about antibiotic resistance among poultry farmers. The study revealed a high prevalence of AMR linked to tetracycline, trimethoprim/sulfamethoxazole, and ampicillin, among others, in these individuals.Oguadinma et al. evaluated cross-tolerance to sanitizer among antibiotic-resistant O157:H7 and non-O157:H7 Shiga toxin-producing E. coli (STEC). This study highlighted a potential public health concern; antibiotic resistance in E. coli could enhance their tolerance to lactic acid and compromise mitigation strategies, such as sanitation, against the pathogen .In their contribution, Kijewska et al. describes the potential of mollusks to become reservoirs of pathogenic bacteria that can carry antibiotic-resistance genes and potentially introduce them into new environments. This finding could expand the existing spectrum of antibiotic-resistance genes present in local biomes .The review article by These contributions provide an overview of current research on AMR in sustainable food systems. They illustrate the multidisciplinary nature of the challenge, requiring collaborative efforts across different sectors and stakeholders, including scientists, policymakers, and industry players.In conclusion, this Research Topic highlights the urgent need to address the challenge of AMR in sustainable food systems to safeguard public health, animal welfare, and environmental sustainability. It calls for a concerted and coordinated effort to promote the responsible and prudent use of antimicrobial agents in all sectors, strengthen surveillance and monitoring systems, develop and implement innovative detection and intervention methods, and raise awareness and education on the risks and impacts of AMR.AC-M, MT, and JE drafted the editorial, participated in discussions about the ideas, and revised the final editorial. All authors contributed to the article and approved the submitted version.


Neuropsychiatric symptoms and syndromes are among the most common non-motor symptoms of Parkinson's Disease but they are frequently unrecognized and untreated. Dopamine Dysregulation Syndrome is an uncommon complication of the treatment of Parkinson's disease, characterized by an addictive use of dopamine far more than the dosage required for treatment of objective motor impairment, leading to severe dyskinesia, euphoria, aggressivity, or psychosis. We present a paradigmatic case of Dopamine Dysregulation Syndrome, Mania, and Compulsive Buying in a 55-year-old male with Parkinson's Disease. We also reviewed the risk factors and the therapeutic management of Dopamine Dysregulation Syndrome in Parkinson's Disease. Dopamine Dysregulation Syndrome (DDS) is an addictive pattern of dopamine replacement therapy use, above the prescribed dosage and those required to control motor symptoms –3. This We present a paradigmatic case of DDS, Mania, and Compulsive Buying in a 55-year-old male with PD.A 55-year-old male with PD was referred to our institution for psychiatric evaluation to undergo Deep Brain Stimulation (DBS) surgery due to debilitating dyskinesias and unpredictable and large periods of “OFF” states. He was diagnosed with PD at 50 years although the first symptoms were reported at age 43. Motor fluctuations and debilitating dyskinesias were reported 2 years before. He also had a history of Depressive Disorder since he was 41 years old. According to the information given by the patient and the family, there is no history in the family of knowing neurologic or psychiatric diseases. At admission, the patient presented dressed in colorful clothes and was wearing three gold necklaces. He presented an elated mood, disinhibition, logorrhea, an increase in speed of speech, increased self-esteem, diminished necessity to sleep, and paranoid delusions. He also displayed impulsive-compulsive behaviors namely compulsive buying and hoarding—he had bought over 5,000 pocket watches and 42 old and unusable cars, that he stored; he also stored old radio devices. He related these periods of excessive buying to periods of higher energy and higher doses of dopaminergic medication. At the time of this appointment, he was medicated with levodopa  and ropinirole 8 mg/daily corresponding to a levodopa equivalent daily dose (LEDD) of 2,310 mg. In a later appointment, he admitted to taking more levodopa pills than those prescribed by the doctor. He maintained the manic symptoms previously described. He also reported waking up in the middle of the night to take dopaminergic pills, associated with an increase in new activities and projects. The patient developed an interest in “old things” and new daily routines. He started leaving home early in the morning and buying old objects. Despite having no insight into these new behaviors, he was admitted voluntarily to the psychiatric ward. During the hospitalization, the dopaminergic medication was gradually reduced , pill taking was controlled and he did not have access to extra pills. Valproic acid and quetiapine extended release were started and increased to 1,000 and 300 mg/daily, respectively and ropinirole was tapered off to 4 mg. The maniac symptoms gradually decreased and there was no worsening of Parkinson's motor symptoms or any change in dyskinesias. He was discharged 29 days after his admission to the psychiatry ward in clinical remission.We provide a clinical description of a paradigmatic case of DDS, Mania, and Compulsive Buying in a patient with Parkinson's Disease. Knowledge about the phenomenology of DDS in patients with PD is still limited and reports of DDS comorbid with other ICDs and Mania are still scarce.punding and impulse control disorders. The use of high doses of levodopa and dopaminergic agonists with short-acting profiles are known risk factors.DDS is an uncommon condition, described for the first time by Giovannoni et al. . The estsubstantia nigra pars compacta (SNc) and in the ventral tegmental area (VTA) with depletion of dopamine in the nigrostriatal motor pathway (nucleus accumbens (NAc) is essential to the reward effect of medication. The over-stimulation of the mesolimbic system can impact the development of rewarding behaviors , which are localized in the mesocorticolimbic system, explaining the risk of developing ICD. Levodopa, especially at higher dosages, was also related with ICD but to a lesser level than DA treatment . HoweverTherapeutic management of DDS is complex and involves gradual tapering or discontinuation of dopaminergic medication, although discontinuation may be associated with severe motor disorders, dysphoria, irritability, depression, and anxiety , 2.Some of the features respond to the minimization of short duration response or reduction of the dosage, while behavioral disturbances often require antipsychotic treatment with clozapine or quetiapine. Other authors previously described the efficacy of valproate in PD patients presenting with DDS, without observing the valproic acid-associated worsening of parkinsonism , 16. ValMania and hypomania in PD are rarely reported besides the immediate post-DBS cases . FactorsThe effort in interrupting the processes of addiction may justify why limiting dopamine replacement therapy or changing to a non-pulsatile formulation can lead to these different symptoms. DBS, by acting in the subthalamic nucleus, can permit a reduction of dopamine replacement therapy and also attenuate the pulsatile nature. However, the use of DBS in resolving DDS has demonstrated ambiguous results, which may be related to the time taken from the start of PD to the use of DBS; less comorbidity, rapid withdrawal of levodopa post-DBS and use of dopamine agonists .Increasing recognition of neuropsychiatric syndromes in PD patients is mandatory to improve treatment which frequently demands a specialized and multidisciplinary team.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the patients/participants or patients/participants legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the individual for the publication of any potentially identifiable images or data included in this article.CS: Writing – original draft, Writing – review & editing. MR: Writing – original draft, Writing – review & editing. IC: Writing – original draft, Writing – review & editing.


Low-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become supply-limited large clusters. In stochastic simulations, such clusters display a wide range of sizes and compositions. We have developed a Python package, MolClustPy, which performs multiple stochastic simulation runs using NFsim (Network-Free stochastic simulator); MolClustPy characterizes and visualizes the distribution of cluster sizes, molecular composition, and bonds across molecular clusters. The statistical analysis offered by MolClustPy is readily applicable to other stochastic simulation software, such as SpringSaLaD and ReaDDy.https://molclustpy.github.io/The software is implemented in Python. A detailed Jupyter notebook is provided to enable convenient running. Code, user guide, and examples are freely available at  Clustering of weakly interacting multivalent proteins and nucleic acids leads to biomolecular condensate formation via phase transition  in a concentration-dependent manner , left toad hoc code to analyze multivalent clustering for different biological systems (Weak molecular interactions result in a distribution of size and compositions of multi-molecular clusters (The user needs to describe the molecules and their interactions in the rule-based BioNetGen Language (BNGL) format  from multiple trials and perform statistical analysis on the combined dataset. A “cluster” is a molecular network where each node is a multivalent molecule, and the bonds comprise the edges. We analyze the network topology and their relative abundances. For example, the degree of a node gives the number of bonds coming out of that particular molecule. BF is then computed as the ratio of bound sites over total sites present in a molecule.We have demonstrated the use of MolClustPy to characterize key properties of molecular clusters derived from multiple stochastic NFsim simulations. A limitation of BioNetGen/NFsim is that it ignores spatial factors that may control cluster growth and composition. These include steric and crowding effects as well as the geometric organization of binding sites. A related spatial issue is the importance of disordered proteins and protein domains in the formation of biomolecular condensates. The SpringSaLaD  or ReaDD


Zea mays var. amylacea and Zea mays var. indurata are maize ecotypes from Paraguay. Aspergillus section Flavi is the main spoilage fungus of maize under storage conditions. Due to its large intraspecific genetic variability, the accurate identification of this fungal taxonomic group is difficult. In the present study, potential mycotoxigenic strains of Aspergillus section Flavi isolated from Z. mays var. indurata and Z. mays var. amylacea that are marketed in the metropolitan region of Asunción were identified by a polyphasic approach. Based on morphological characters, 211 isolates were confirmed to belong to Aspergillus section Flavi. A subset of 92 strains was identified as Aspergillus flavus by mass spectrometry MALDI-TOF and the strains were classified by MALDI-TOF MS into chemotypes based on their aflatoxins and cyclopiazonic acid production. According to the partial sequencing of ITS and CaM genes, a representative subset of 38 A. flavus strains was confirmed. Overall, 75 A. flavus strains (86%) were characterized as producers of aflatoxins. The co-occurrence of at least two mycotoxins  was detected for five of the Z. mays samples (63%). Considering the high mycological bioburden and mycotoxin contamination, maize marketed in the metropolitan region of Asunción constitutes a potential risk to food safety and public health and requires control measures. Zea mays L.) is one of the most important grains in the world . After air-drying, mass spectra were obtained using the MALDI-TOF MS Autoflex Speed , which was equipped with a smart beam laser source (355 nm). All samples were analyzed in triplicate.Each sample (1 μL) was transferred into the MALDI sample plate  and, once dried, 1 μL of matrix solution was added + or forming adducts with Na+ or K+ cations (cationization) were detected  software was used to compare the obtained spectra with the spectra of the following standards: aflatoxin B1 (C.391 Da)  16,42].,42.17H12detected .Z. mays var. indurata (locro) or Z. mays var. amylacea (avatí-morotĩ). Mycotoxin content was analyzed as follows: total aflatoxins (AFs) [® from Romer Labs .The mycotoxin content in the maize samples was determined by direct competitive enzyme-linked immunosorbent assays (ELISA). All samples picked from the same location were mixed according to the type of maize: ns (AFs) , total fns (AFs) , total zns (AFs) , and T-2ns (AFs) . This wav/v) solution and the procedure was continued following the manufacturer’s recommendations. Optical densities (ODs) were determined using the microwell strip reader at 450 nm in a MultiSkanTM FC  system. The samples’ ODs were compared with the ODs of the standards and the obtained concentrations were determined in ppb (μg.kg−1).Each maize sample (20 g) was milled with a grain mill. An extraction of samples was made with a methanol:water (70:30 p < 0.05) using the GraphPad Prism 8.0.0. software for Windows, San Diego, CA, USA, www.graphpad.com. A cluster analysis was performed and a dendrogram of phenotypic relatedness was constructed using the IBM® SPSS® Statistics software for Windows 25.0.The mean fungal frequencies and the mean aflatoxin concentrations were subjected to a three-way ANOVA (multivariate) test, followed by Tukey’s HSD test  was performed using GenAlEx® 6.5 software [Aspergillus flavus isolates were selected by a random sampling method based on morphologically and physiologically characterized isolates representing the phenotypes of Aspergillus flavus (section Flavi), Aspergillus tamarii (section Flavi), A. awamorii (section Nigri), and Aspergillus section Aspergillus.To evaluate the association between aflatoxin production and the sclerotia morphotype, a logistic regression analysis was performed using the IBMsoftware . AspergiAspergillus, Fusarium, and Penicillium. After a preliminary identification based on morphological characters, 291 isolates were confirmed as belonging to the genus Aspergillus. A total of 211 out of 291 fungal strains were identified as Aspergillus section Flavi, the most predominant taxonomic group within the Aspergillus group (72%). Other species were distributed within Aspergillus section Aspergillus (21%), Aspergillus section Nigri (5%), and Aspergillus section Circumdati (2%).A total of 521 fungal isolates were obtained from assessed maize samples. The predominant genera were Z. mays var. amylacea samples, which were contaminated with strains of Aspergillus spp., Fusarium spp., and Penicillium spp. In the samples of Z. mays var. indurata, Aspergillus spp. was the main contaminant while Penicillium spp. was isolated at low frequency. No strains of Fusarium spp. were isolated from Z. mays indurata samples.The highest fungal abundance was observed for the Z. mays variety was different among the local markets. A similar diversity of fungal genera was observed in Z. mays var. amylacea from SP2, SP4, and SP5. In SP1, Z. mays varieties were more contaminated with Aspergillus than samples from other sampling points were. In SP3, the frequency of Fusarium spp. in the Z. mays var. amylacea samples was significantly higher (ca. 45%) compared to that of other SPs  produced L-type sclerotia (≥400 µm) and 13% (n = 28) produced S-type sclerotia (<400 µm). The other 17% of the isolates (n = 36) did not produce sclerotia.The sclerotia production was evaluated for the 211 strains previously identified by their morphological traits as A. tamarii (section Flavi), presented brown colonies and rough conidia. Isolates classified as A. awamori (section Nigri), presented dark brown/black colonies and pale yellow reverse colonies. In CYA media, cultures presented black colonies with white edges; meanwhile, on MEA media, cultures were grayish. The conidia were lightly rough to rough. Isolates classified as morphotype Aspergillus section Aspergillus, showed no growth in the CYA at 37 °C and showed limited growth in both the CYA at 25 °C and the MEA. In terms of the micro-morphology, 60% of the isolates showed the presence of ascospores; the other 40% showed the anamorphic phase with uniseriate conidiophores and conidia.Isolates classified as Aspergillus strains, from species such as Aspergillus tamarii (section Flavi), A. awamori (section Nigri), and Aspergillus section Aspergillus, were used as outgroups in the principal coordinate analysis (PCoA)  . Accordiulations .Aspergillus section Flavi strains were identified, based on the proteomic profiles created by MALDI-TOF MS, as A. flavus  and calmodulin (CaM) genes. The phylogenetic trees were constructed based on the calmodulin (CaM or CMD5) gene sequences via two inference methods: maximum likelihood and Bayesian inference (A representative subset of 38 nference . Both anA. flavus isolated from Paraguayan maize and the reference samples from different regions of the world (A haplotype network based on the CaM sequences (545 bp) was elaborated to evaluate the existence of a phylogeographic association among the he world . Thirteehe world . Each ciA. flavus isolates analyzed showed an association with those found in databases from other countries or regions, such as Argentina, Eastern Europe, Indonesia, Madagascar, Nigeria, Paraguay, Republic of Korea, Thailand, and the United Kingdom; these corresponded with haplotype I. Haplotype I is a dominant haplotype with a ubiquitous geographical distribution. For haplotype II, the isolates from Paraguay showed an association with samples from Argentina, Brazil, Eastern Europe, The Netherlands, and Republic of Korea. No mutational steps were observed, suggesting that they were a frequent haplotype. In addition, considering the distance and lower frequency, haplotypes IV and V (exclusively isolated from Paraguay), have the characteristics of new haplotypes.Most of the A. flavus, previously identified by MALDI-TOF MS, were evaluated. Only 36 out of 83 isolates presented bands corresponding to the four genes, aflD, aflM, aflP, and aflR. Data obtained showed that under tested conditions, 86% of fungal strains (n = 31) produced aflatoxins and 14% (n = 5) were not aflatoxin producers  (All 92 id (CPA) .A. flavus isolates were grouped according to their chemotypes, as shown in Aspergillus flavus isolates presented a variable number of peaks in their mass spectra, according to their ability to produce other kinds of metabolites.Based on the mass spectra obtained, the n = 92); although, no statistical significance was observed (p > 0.05).The chemotypes VI (AFG) and VII (AFB + AFG) were predominant, being observed for 68% of the isolates evaluated. These were followed by chemotypes III (AFB) and V (non-aflatoxin producers), being associated with 19% and 14% of isolates, respectively. The remaining isolates were distributed between chemotypes II  and I  . A uniquZ. mays var. amylacea maize, as determined by ELISA, showed a contamination range spanning from 1.67 ± 0.2 to 20.75 ± 0.6 µg·kg-1. For the Z. mays var. indurata from SP1 and SP2, total AF concentrations were below the limit of detection (LOD < 1 µg.kg−1). Meanwhile, from SP3, the total AF concentration was observed as 2.30 ± 0.7 µg.kg−1 (In  µg.kg−1 .-1). For ZEA, the obtained concentrations ranged from 21.64 ± 1.6 to 30.09 ± 3.0 µg.kg−1 in Z. mays var. amylacea and from 22.69 ± 2.0 to 247 ± 6.7 µg.kg- in Z. mays var. indurata. Regarding FUM, all of the samples of Z. mays var. indurata analyzed showed a FUM concentration below the LOD (200 µg.kg−1). In Z. mays var. amylacea, the FUM concentrations ranged from 329.65 ± 36.8 to 1782.81 ± 271.0 µg.kg−1, except for the samples from SP4, which showed values below the LOD. The co-occurrence of at least two mycotoxins  was detected in 71% of the samples analyzed.For the T2 toxin, all samples analyzed showed values below the LOD ; there was variation among these three SPs. Considering only the results above the LOD, the Z. mays var. amylacea from SP1 showed the highest concentration of total AF; meanwhile, the lowest total AF concentrations were observed in samples from SP4. Samples from SP3 were excluded from the analysis because their concentrations were below the LOD.The ANOVA performed with the total AF concentrations, in terms of the samples of Z. mays native germplasm. This agricultural product is intrinsically associated with the Paraguayan culture, being initially used as a nutritional source and raw material for the ceremonial drinks of native communities. Until now, maize remains an important staple food for the Paraguayan population. Despite this, the food safety of Paraguayan maize varieties has been poorly considered.Paraguay displays a pool of Z. mays var. indurata and Z. mays var. amylacea was performed. The Aspergillus spp. and Fusarium spp. show the highest incidence in Z. mays var. amylacea. Conversely, in Z. mays var. indurata, only Aspergillus spp. was dominant.In the present study, an analysis of the mycological bioburden of Aspergillus population in maize from Argentina. The authors reported that A. flavus (n = 752) was the most abundant strain. Moreover, the authors reported significant isolation of Fusarium sp., followed at a lower frequency by the Penicillium, Rhizopus, and Absidia species [Fusarium, Penicillium, and Aspergillus glaucus groups were the major contaminating genera in genetically modified (GM) and non-GM maize kernels samples from Brazil.Sepúlveda et al.  analyzed species . Similar species  reportedZ. mays var. indurata presented less fungi contamination. This possibly occurred due to the physical process that maize is subjected to before its commercialization. In this physical process, the pericarp is excluded, eliminating part of the field contamination. Furthermore, the Z. mays var. indurata is a flint-type corn with a hard endosperm, unlike Z. mays var. amylacea, which shows a very soft endosperm. The hard endosperm could provide a mechanical barrier to spoilage fungi damage [Regarding the maize variety analyzed in the present study, i damage ,49.Aspergillus section Flavi species. Quéro et al. [Aspergillus section Flavi. The authors reported the correct identification of 99.65% of the fungal strains.Concerning the correct identification of food-contaminating fungi, in the present study, MALDI-TOF MS proved to be helpful in the identification of the o et al.  used MALAspergillus section Flavi by morphological methods, which was not sufficient for species identification. Therefore, MALDI-TOF MS analysis was used as an additional tool. Herein, four clades were observed in the dendrogram obtained from the MALDI-TOF mass spectra results. These clades were not completely related to the phenotypic profile; consequently, it was not possible to differentiate aflatoxigenic isolates from non-aflatoxigenic isolates. These results are corroborated by previous studies in which the separation of aflatoxigenic isolates from non-aflatoxigenic isolates was not achieved by MALDI-TOF MS [In the present study, 211 fungal strains were previously classified as I-TOF MS .Aspergillus section Flavi isolates obtained from a market in Lavras, Brazil. The MALDI-TOF MS analysis was not sufficient enough to differentiate aflatoxigenic from non-aflatoxigenic Aspergillus. However, the authors reported that in a polyphasic approach, the proteomic profile provided by MALDI-TOF MS, when associated with classical methodology, increased the reliability of the results.Similarly, Silva et al. 2015  used theAspergillus section Flavi [Aspergillus section Flavi autochthonous strains isolated from diverse substrates. Likewise, the MALDI-TOF mass spectra obtained in the present work can be used to supplement MALDI-TOF MS databases by adding isolates from maize of Paraguay, which has its own characteristics and genetic variability.Considering the above, MALDI-TOF mass spectrometry is a powerful tool to differentiate closely related fungal species. Additionally, a polyphasic approach is recommended for the identification of cryptic fungal species, such as on Flavi ,50,51,52on Flavi ,54,55,56Aspergillus spp. is based on the amplification and partial sequencing of the ITS and CaM, or other genes, such as beta-tubulin and RPB2 (RNA polymerase II second largest subunit) [As mentioned above, for cryptic species, a comprehensive analysis including the results obtained from morphological, physiological, and molecular methods (polyphasic approach) was necessary ,57. The subunit) ,58,59.Aspergillus section Flavi isolates. In the present study, the identification of 38 Aspergillus flavus is supported by molecular biology information. The molecular biology data were used to support the previous identification based on morphology and MALDI-TOF MS analyses.Combining the information obtained from ITS and CaM sequencing is pivotal for the identification of the A. flavus isolates from different geographical regions, such as Argentina, Eastern Europe, Indonesia, Nigeria, South Korea, Thailand, and the United Kingdom. However, three haplotypes  were more distant from the others, suggesting that these may be exclusively from Paraguay [In addition, with the haplotype network analysis, the phylogenic data confirmed that most of the isolates were similar, in terms of the CaM gene, to Paraguay .A. flavus in maize used for feeding humans and livestock.In addition to the identification achieved by the polyphasic approach, in the present work, an analysis of physiological characteristics was performed. Physiological characteristics are relevant for the comprehension of biodiversity populations of Fusarium FUM-producers’ strains (16).MALDI-TOF mass spectrometry is a highly sensitive technique used to detect organic molecules present in low concentrations in different kinds of matrices. MALDI-TOF MS can be used to detect fungal secondary metabolites ,62 and hn = 79) of the A. flavus strains were characterized by MALDI-TOF MS as producers of aflatoxins. The analyzed isolates were categorized into eight chemotypes based on the classification of Vaamonde et al. [A. flavus strains.In the present study, 86% , causing immunotoxic and cytotoxic effects in end consumers [Cyclopiazonic acid is the main contaminant of Kodo millet grain ; but, in wheat and soybean, this was less frequent  [The data presented here showed that the frequency of CPA-producing strains was lower than what was shown in the results found by Vaamonde et al. . Accordictively) . Other tA. flavus population [A. flavus has increased since exclusion by competition has been shown to be an efficient way of reducing aflatoxin levels in maize [According to Olarte et al. , the clupulation . The seain maize ,71,72. Sin maize ,74.A. flavus population is the sclerotia. In the present study, 83% of A. flavus isolates produced sclerotia at 30 °C, a common room temperature in Paraguay (average of 31.5–34.9 °C to 20.1–23.7 °C) [A. flavus has a great potential to spread and survive in this agro-ecosystem [Another well-studied characteristic of the 23.7 °C) . This pocosystem ,64.A. flavus strains was L-type (70%), >400µm. Similarly, Rocha et al. [A. flavus being isolated from maize samples (n = 200) from different geographical regions and climate conditions in Brazil. All isolates were classified as sclerotia L-type [A. flavus were isolated in high frequency (98.5%) and all of them were sclerotia L-type. Probst et al. [A. flavus type L.The predominant sclerotia among the analyzed a et al.  reporteda L-type . Likewisa L-type  analyzedt et al.  evaluateAspergillus species from soil, stubble, and insect samples from maize fields in three periods: pre-sowing, in-crop, and post-harvest. The frequency of A. flavus sclerotia producers ranged from 18 to 34%. However, all of the isolates that produced sclerotia were L-type.Nesci and Etchevery  isolatedA. flavus strains, all L-type, from maize samples from five districts in Northern Italy. According to the authors, A. flavus type L is the causal agent of aflatoxin contamination in maize in Italy [According to Bayman and Cotty , due to in Italy . Similarin Italy  reportedin Italy ,83,84,85A. flavus strains isolated in Paraguay that produce both aflatoxins B and G. Until now, no correlation between sclerotia morphology and AF biosynthesis could be determined. Different studies have pointed out that AF production is related to multifactorial factors, such as genotypes, climate changes, and agricultural practices [The present study is the first report on S- and L-type ractices ,85,86,87A. flavus isolates with different morphological characteristics, such as colony color, sclerotia type , and physiological markers , forming different phenotypic groups. The data presented herein corroborate the findings of previous studies [A. flavus population is phenotypically diverse.In the present study, PCoA grouped  studies ,88, whicA. flavus isolates were amplified for AF genes  and produced AFs. Conversely, the AF biosynthesis was not detected in five A. flavus strains; although, it amplified the four AF genes.Regarding the mycotoxigenic characterization conducted by using molecular biology, 31 aflD, aflM, aflO, aflP, aflQ, aflR, and aflS, from A. flavus maize isolates from Italy using a PCR and determined four amplification patterns. They reported the presence of isolates that amplified all seven genes but did not produce aflatoxins. Oloo et al. [A. flavus isolates (n = 72). Among the seven AF biosynthesis genes assessed , only the presence of aflD and aflS significantly increased the amount of total aflatoxin production.Gallo et al.  analyzedo et al.  analyzedA. flavus is genetically diverse. Nevertheless, works such as the present study are useful for determining the strains responsible for contaminating foods and characterizing isolates that can be used in terms of biological control [Based on the literature, the population of aflatoxin-producing and non-producing  control ,76,91.Z. mays var. amylacea detected with high contamination levels of Aspergillus and Fusarium strains also showed high concentrations of AF, FUM, and ZEA.The presence of mycotoxin-producer fungi in maize is considered a primary indicator of mycotoxin contamination. This correlation was confirmed in the present study. The samples of Z. mays var. indurate, ZEA was the most prevalent mycotoxin. The detection of ZEA, despite the absence of Fusarium strains in Z. mays var. indurata, suggests that fungal infection, as well as mycotoxin contamination, occurs mainly in the field [Fusarium sp. can also be present in stored grains, even though over time, this is shifted by the interaction with the mycobiota and by the decrease in relative humidity [Regarding he field ,15. It hhumidity ,92.−1 and 2.0 µg.kg−1, respectively [The European Commission (EC) established rigorous legislation for mycotoxins in maize products, considering maize’s different processing and derivative products. For total AFs (AFB1 + AFB2 + AFG1 + AFG2) and for AFB1 in all cereals and products derived from cereals, including maize products, the EC has set the maximum allowed levels (MAL) at 4 µg.kgectively .−1. For fumonisins (sum of FB1 and FB2), the MAL has been set at 1000 µg.kg−1. The base standard for MERCOSUR countries regarding AFs in maize is the technical regulation on maximum limits of aflatoxins MERCOSUR/GMC/RES N°. 56/94 [−1) in maize grain and its derivatives to be five times higher than those set by the EC. In addition, no MAL was established for the Fusarium toxins  in maize products [In addition, the EC has set the MAL of ZEA in maize, for direct human consumption, maize-based snacks, and maize-based breakfast cereals, at 100 µg.kgZ. mays var. amylacea from SP1 (20.75 ± 0.6 µg.kg1) and SP5 (6.65 ± 1.5 µg.kg−1) presented a total AF concentration higher than the limits established by the EC. Additionally, a sample of Z. mays var. amylacea from SP1 also exceeded the MAL of FUM (1782.81 ± 271.0 µg.kg−1).In the present study, two samples of Z. mays var. indurate, one sample (247 ± 6.7 µg.kg−1) exceeded the MAL for ZEA. Zearalenone is not carcinogenic but it has a potent estrogenic effect in animals, especially in pigs [For  in pigs . In humaDue to the popular belief that it increases milk production, the intake of maize-based foods is higher among Paraguayan pregnant and puerperal individuals. In addition, maize-based foods are also part of the schools’ snack menus in Paraguay .Previous studies have reported mycotoxin co-contamination in maize samples worldwide ,98,99. HA. flavus population and the co-occurrence of aflatoxins, fumonisins, and zearalenone in the native Paraguayan maize commercialized in the local markets. In such local markets of Paraguay, maize is stored in improper conditions, piled up in burlap bags with no control over the temperature and humidity. The inadequate storage of maize can promote the growth of Aspergillus, Fusarium, and Penicillium strains, which grow in a wide range of temperatures and water activities (aw) [The present study contributes to the understanding of the biodiversity of the ies (aw) ,104,105.Aspergillus section Flavi characterization since this section includes the main aflatoxin producers [To mitigate spoilage fungi and mycotoxin contamination in Paraguayan maize products, a combined strategy should be applied; as management strategies, the use of a tolerant germplasm, good agronomic practices, and good manufacturing practices could be included. Other than that, mycobiota characterization is a key element that should be used to mitigate mycotoxin contamination. Regarding this, the analysis presented herein is focused on the roducers ,112,113.Aspergillus section Flavi strains from Zea mays var. amylacea and Zea mays var. indurata, two maize ecotypes from Paraguay. Species of Aspergillus and Fusarium showed the highest incidence in Z. mays var. amylacea. In Z. mays var. indurata, species of Aspergillus were dominant.This study provides the first comprehensive dataset regarding the genetic diversity and aflatoxigenic potential of Z. mays var. amylacea. Meanwhile, in Z. mays var. indurata, ZEA was detected in all samples. Despite the absence of the Fusarium strains, the detection of ZEA suggests that the field is a critical point for fungal infection and mycotoxin contamination during Z. mays var. indurata production. The detection of AF, FUM, and ZEA and the high mycological burden emphasizes the need for improvements regarding the Zea mays production chain.Regarding mycotoxin contamination, AF, FUM, and ZEA were detected in one sample of SP1 and one sample of SP5 in Aspergillus section Flavi. More studies covering different agro-ecological zones and seasons are needed for better comparisons to be made among the mycotoxigenic mycobiota of Paraguayan Zea mays.In the present work, the fungi identification conducted using a polyphasic approach  proved to be efficient for identifying and discriminating closely related species encompassed in


In an earlier paper from 2019, this author concluded that successful eradication of bovine tuberculosis (bTB) from Ireland by 2030 would be unlikely, given control strategies in place at that time plus the addition of badger vaccination. He argued that additional measures will be needed, broadly focusing on bTB risks from wildlife, risk-based cattle controls, and industry commitment. This paper considers these points in further detail.Ongoing monitoring of the badger vaccination programme  and associated research will be critical, with a focus both on programme inputs and outcomes. The direct contribution of cattle movements to bTB restrictions in Ireland has been evaluated. However, it is the indirect role of cattle movements in bTB restrictions that is likely of greater importance, particularly towards the latter phase of the eradication programme. In other national programmes, a range of risk-based approaches have been used to address the challenge of residual infection in cattle , and similar approaches are needed in Ireland. A number of authors have highlighted the critical importance of industry commitment to programme success, and the key role of programme governance to achieving this. In this commentary, the author briefly considers experiences from Australia and New Zealand in this regard. The author also reflects on the challenge of uncertainty in decision-making, the relevance to Ireland of lessons from other countries, and the potential contribution of new methodologies in support of the national programme.‘The tragedy of the horizon’ was a term first used in the context of climate change, referring to the costs imposed on future generations that the current generation has no direct incentives to fix. This concept is equally relevant to bTB eradication in Ireland, where current decisions will have long-term consequences for future generations, including both the general public (through the Exchequer) and future Irish farmers. In recent years, there has been a deterioration to the national bovine tuberculosis (bTB) situation in Ireland. From an historic low herd incidence rate of 3.27% in 2016, this metric has risen to 4.33% in 2021, and 4.31% in 2022. Concurrently, total expenditure of the programme has increased from €82 million in 2015 to €97 million in 2020, with the Exchequer being the largest contributor  [M. bovis conference that was held in Galway, Ireland, in June 2022. This commentary briefly considers these points in further detail.In an earlier review , I conclCervis nippon, an introduced species, and Sika hybrids) are a bTB maintenance host. In Co. Wicklow, however, and potentially elsewhere, this may change , due to the influence of those factors that facilitate the establishment and perpetuation of bTB in deer. Ongoing ecological and epidemiological research in deer will be important.Wildlife play a role in the epidemiology of bTB in cattle in many countries, and represent a key challenge to eradication efforts. In Ireland, there is robust evidence in support of the efficacy of field badger vaccination by injection, and – with uncertainty – of the positive contribution of badger vaccination to national eradication efforts. For some years now, there has been a national roll-out of a badger vaccination programme, seeking to progressively replace badger culling. Ongoing monitoring and research will be critical, both of programme inputs  and outcomes , and to determine reasons for success and failure. In most areas of Ireland, there has been no evidence to date to suggest that deer  is a recognised feature of bTB epidemiology, and a further challenge to eradication. This phenomenon occurs within test-based eradication programmes throughout the world, and is the key rationale for ongoing efforts towards improved diagnostic tests. Because there is very substantial cattle movement in Ireland, there is no doubt that ongoing commerce will result in the movement of some cattle with residual infection. This has been evaluated in a range of studies, including an estimate of 7.4% of bTB restrictions that are directly attributable to the recent introduction of an infected animal . HoweverA number of authors have highlighted the critical importance of industry commitment to programme success, and the key role of programme governance to achieving this. In Australia, fundamental change to programme governance were introduced in 1984  coinciding with rising industry opposition to the use of mass destocking to tackle bTB (and brucellosis) in difficult northern areas. With these changes, industry subsequently played a central role in programme decision-making at all levels. The trust built between government and industry during this and associated programmes, and the lessons learned, has played a key role in the establishment and ongoing operation of animal health programmes in Australia, including Animal Health Australia. In New Zealand, TBfree has been the statutory management agency responsible for bTB eradication since 2013. Working in partnership with government and the livestock industries, TBfree operates within a defined cost envelope that encompasses all programme costs, including compensation. A legally agreed costing model is in place, underpinned by national legislation, based on formal recognition of beneficiaries (those who benefit from bTB control) and exacerbators . Strategic planning, robust science and programme success are each recognised as the key means to mitigate the financial liabilities borne by industry (and to a lesser extent government). This partnership model, in place in New Zealand since 1993, has overseen substantial and sustained progress towards bTB eradication. Although there is now greater stakeholder involvement in Ireland following the establishment of the national TB Forum in 2018, programme governance remains firmly under the remit of government. Further, current cost- and responsibility-sharing arrangements in Ireland do not accurately reflect the balance of private goods  and public goods  that derive from bTB eradication, as determined in a recent national review [per se is rarely justification for inaction. Here, we need to distinguish uncertainties that have the potential to lead to a fundamental – compared to an incremental – shift in knowledge about bTB epidemiology and eradication options. To illustrate, there is currently considerable uncertainty about the relative contribution of different infection sources , and an improved national understanding will certainly be helpful . However, this knowledge, once available, is unlikely to challenge the principle – accepted internationally – that all sustaining infection sources must concurrently be addressed for national bTB eradication efforts to be effective.Firstly, decision-makers need to take care when dealing with uncertainty  in the context of bTB eradication. Decisions will always be made in the context of some degree of uncertainty, and it is for this reason that uncertainty Secondly, there has been a view that lessons from other countries cannot readily be extrapolated to Ireland, given differences in farming systems, climate, wildlife hosts etc. Certainly there are differences; however, it is also clear that the broad principles of bTB eradication have been similar across all environments where bTB has proved (or is proving) successful. This is particularly the case with respect to cattle controls and industry commitment. For example, no country has yet been able to eradicate bTB without introducing comprehensive, risk-based controls on cattle movement.Thirdly, a range of promising new methodologies are now emerging, including whole genome sequencing, mathematical modelling and machine learning. These are now being implemented in Ireland, and have the potential to provide greater clarity in current areas of scientific uncertainty.I highlight three key points relevant to the issues raised previously:In the current agri-political environment in Ireland, it can be very challenging to make difficult or unpopular decisions. Given existing costing arrangements and the current national bTB trajectory, in part this is because decisions made now may have only a limited positive impact on the current generation of Irish farmers, certainly over the shorter term. We are constrained therefore to those decisions that are politically possible. However, with reference to bTB eradication, current decisions will have long-term consequences for future generations, including both the general public (through the Exchequer) and future Irish farmers. This is because these decisions will influence both ‘time to eradication’ and overall programme costs . This concept, called ‘the tragedy of the horizon’ was first used by the Governor of the Bank of England in the context of climate change , and ref