Lighting up the relevant lesion boundaries during operations is vital for guiding the effective resection of hepatopathic tissue. IAN) were prepared and evaluated as molecular-logic gates. The input and output signals of the IAN derivatives were successfully used to highlight different hepatopathic regions in order to facilitate boundary differentiation. The IAN derivatives produce different signals due to collaborative changes in the conformation and structure. The hepatopathy-related enzymes (COX-2 and NAT) were used to induce conformational and structural changes in IAN derivatives. Based on these enzyme induced synergistic effects, IAN can sensitively emit different coloured signals such as green, cyan and blue  as a function of the different input signals, i.e. the different activity of COX-2 and NAT in solution and living cells. Significantly, the IAN derivatives were successfully used to distinguish the boundaries of hepatopathic lesions in tissues after spraying with IAN derivatives  under a hand held lamp at 365 nm by naked eye.Lighting up the relevant lesion boundaries during operations is vital for guiding the effective resection of hepatopathic tissue. We envisioned that molecular-logic gates, which are known for their excellent digital correlation between input and output signals, could be used to facilitate differential visualization of lesion boundaries. Herein, a series of flexible molecules, naphthalene imide-indole derivatives ( Hepatoma can evolve from the severe deterioration of hepatitis and cirrhosis, and results in a high death rate.Based on this strategy, many excellent organic molecules have been designed to monitor biological enzymes involved in the deterioration process of hepatopathy.IAN derivatives) were designed and synthesized in this work, which display collaborative changes in molecular conformation and structure on exposure to COX-2 and NAT. IAN derivatives display a conformational change on exposure to COX-2 resulting in emission at 530 nm, in contrast exposure to NAT results in a structural change and emission at 490 nm. Importantly, IAN derivatives display good linear response relationships towards increasing activities of COX-2 and NAT. Therefore, the IAN derivatives could emit different outputs depending on the activities of COX-2 and NAT. More importantly, such output signals produce clear logic responses for the different stages in liver disease deterioration. Indicating that our molecular-logic IAN derivatives could be used to monitor and differentiate cirrhosis and hepatoma, which in turn could potentially be developed into a system capable of distinguishing the boundary of different lesions during surgery.With this in mind, a series of the flexible organic molecules, naphthalene imide-indole derivatives , different high-contrast visual signals are needed to distinguish them from the mild lesions and healthy tissue. Such specific clinical demand sets a considerable design challenge for the development of efficient imaging agents (molecular-logic gates). We propose that to achieve this target, we need to uncover new mechanisms of action and recognition targets.IAN derivatives) were designed and synthesized, with which to monitor COX-2 and NAT through changes of the molecular structure and conformation. In the molecular design, 1,8-naphthalimide . Then, indomethacin  were investigated in PBS buffer solution. In the absence of COX-2 and NAT in the test system, 3IAN2 and 4IAN2 emitted at about 530 nm when excited at 430 nm , which is much lower than the previously reported values.The fluorescence changes for the  S13IAN4 , 3IAN6  energies of the optimized conformations (Table S23IAN4 exists predominately in a stable folded conformation (i.e.3IAN4-Folded) in PBS buffer solution, since the FMO energy of 3IAN4-Folded was 8.3 kcal mol–1  within the hydrophobic cavity of COX-2 due to size constraints and multiple hydrogen bonds, the binding affinity of 3IAN4 to COX-2 was found to be 56.33 kcal mol–1  all exhibited the same behavior as 3IAN4 due to similar conformational changes. Conversely, according to their FMO energies  with NAT were investigated in PBS buffer solution. 3IAN2, 3IAN4, 3IAN6, 4IAN2, 4IAN4 and 4IAN6 produce similar signal outputs towards NAT. When NAT was available in the test system, the intensity . Importantly, the time between the two recognition events is significantly shortened to about 150 s compared with previously reported work,3IAN4 is suitable for the imaging of enzymes in living biological systems. 1H NMR titration  of 3IAN4-Unfolded is lower than that of 3IAO4-Unfolded  has transformed into 3IAO4-Unfolded (blue fluorescence) in the presence of NAT and COX-2. And some of 3IAN4-Folded (no fluorescence) has transformed into 3IAO4-Folded (no fluorescence) in the presence of NAT (iv) When there is only the activity of NAT in the system, 3IAN4 produces no emission signal . Furthermore, the molecular dynamic results indicate that the binding free energy of 3IAN4 and NAT in the absence of COX-2 is –42.54 kcal mol–1  Ae signal , which in signal  under thal mol–1  and the M Fig. S8. These dIAN derivatives . i.e. only a molecular conformation change. Therefore, 3IAN4 as molecular-logic gate could output different signals to distinguish the different stages of the hepatopathic samples by microscopic imaging due to the differing activity levels of COX-2 and NAT in the samples. Furthermore, 3IAN4 could distinguish the boundaries of different stages of the hepatopathic samples by naked eye using a hand held lamp , severe cirrhosis , early liver cancer  and advanced liver cancer , respectively. Furthermore, the parallel results . Consequently, this novel IAN derivative could potentially provide a visual tool to help distinguish relevant lesion boundaries during surgery and improve the success rate of hepatopathic resection operations.In conclusion, based on the clinical requirement to light up the relevant lesion boundaries during operations, we have designed and synthesized a series of novel The authors declare no competing financial interests.Supplementary informationClick here for additional data file.


Kallmann syndrome (KS) is a rare inherited genetic disorder characterized by hypogonadotropic hypogonadism and hyposmia/anosmia. Early diagnosis is the key to timely treatment and improvement of prognosis in patients with KS. As the most common complication of KS, renal agenesis can provide clues to early diagnosis and treatment for KS. In this article, we report a case of KS with 8 rare urinary disorders for the first time.A 19-year-old Chinese man presented with 8 rare urinary disorders and a history of bilateral cryptorchidism came to us for micropenis, hyposmia, and delayed puberty.KAL1) gene in exon11, c.1600G > A, p. Val534Ile; 2) Prokineticin receptor 2 (PROKR2) gene in exon 2, c.533G > A, p. Trp178Ser. which led to a diagnosis of KS.The patient presented with hyposmia, low levels of sex hormones and showed a weak response to the GnRH stimulation test leading to a diagnosis of KS. Two missense mutations were found in further whole-exome sequencing: 1) Kallmann syndrome 1 (The patient underwent replacement therapy of human chorionic gonadotropin (HCG) and human menopausal gonadotropin (HMG). The patient had previously undergone six surgeries for cryptorchidism and urinary disorders.The patient's puberty retardation was effectively alleviated. His serum testosterone (T) reached a normal level (8.280 nmol/mL). During the follow-up period, he presented with Tanner stage II pubic hair development.KAL1 and PROKR2 in a case of KS. Among them, bilateral giant kidneys, urinary extravasation of right renal, bilateral megalo-ureters, left ureteral terminal obstruction, bilateral renal cyst and bladder emptying disorder are reported for the first time, which enrich the integrity of urinary disorder types and provide clues to genetic counseling in patients with KS.In this article, we report 8 rare urinary disorders with missense mutations of  These genes appear to involve in the migration of a group of neurons specialized in the sense of smell as well as neurons producing gonadotropin-releasing hormone (GnRH). KS may present as either a sporadic or a familial case, which contains 3 modes of inheritance: autosomal dominant, autosomal recessive, and X-linked recessive inheritance. The relationship between clinical phenotypes and gene mutations may provide clues for early diagnosis and therapy. Renal agenesis is considered as a signature phenotype of KAL1 mutations and can be used as an early marker for genetic screening. In this article, we for the first time reported a diagnosis of KS in a 19-year-old young man with 8 rare urinary disorders demonstrating a conservative change in the KAL1 gene  and the PROKR2 gene  in this article.Kallmann syndrome (KS) is a rare inherited genetic disorder characterized by hypogonadotropic hypogonadism and hyposmia/anosmia.22). At the age of 15, no pubertal development (no pubic hair) was noted. His penis was 2 cm in length and testicles were both 1.5 ml in volume. He had decreased levels of serum follicle-stimulating hormone , luteinizing hormone , and T . His progesterone (P) level was elevated . The diagnosis of hyposmia was confirmed by the olfactory test, showing the ability to distinguish alcohol, water, and vinegar, but not weak amyctic substances such as coffee and bread. The results of pituitary MRI showed that there was no abnormal signal, and the pituitary gland was slightly flattened. The patient was suspected of having KS, and therefore a GnRH stimulation test  was performed. FSH and LH levels at 0, 30, 60, and 90 minutes after GnRH injection were tested and the levels of FSH were 0.860, 2.250, 2.950, 3.040 mIU/ml and the levels of LH were 0.400, 0.990, 1.020, 0.860 mIU/ml, respectively. The patient underwent whole-exome sequencing, which identified two genetic defects:The patient is a 19-year-old Chinese male who presented to our hospital in 2015 for delayed puberty and experienced hyposmia for 3 years. The patient declared that he had experienced hyposmia for 3 years, but he ignored the symptom due to a history of nasitis. There is no relevant history in his family. In 2005, he accepted bilateral testicular traction and fixation for bilateral cryptorchidism, and received HCG (doses unknown) injection after the operation. Then, he was diagnosed with a complex of urinary dysfunctions, including bilateral giant kidneys and hydronephrosis, traumatic rupture of right renal, urinary extravasation of right renal, bilateral megalo-ureters, left ureteral terminal obstruction, bilateral renal cyst, and bladder emptying disorder. The patient underwent 5 operations for some of the above urinary disorders (Table 1)KAL1 gene: a G to A transition at position1600 in exon 11, changing codon 534 encoding valine into isoleucine;a missense mutation in the 2)PROKR2 gene, a G to A transition at position 533 in exon2, changing codon 178 encoding tryptophane into serine  and CHARGE syndrome, on the other hand, were also considered and precluded.After 2 years of HCG and human menopausal gonadotropin (HMG) replacement therapy, his serum T reached normal level and Tanner stage II pubic hair development was observed. The details of hormone replacement therapy (HRT) and outcomes were showed in Table 3–6 The main causes of KS are the impairment of olfactory axis development and of GnRH neurons migration caused by gene mutations. The primary purpose of treatment for male patients is to promote masculine development, and the second is to restore the gonadal function and even fertility of patients who are eager to give birth.According to the literature, the incidence of KS ranges from 1/10,000 in males and 1/50,000 in females, which is 5 times higher in males than in females. renal malrotation, bilateral dilatation of the calyces and pelves, horseshoe kidneys, multicystic dysplastic kidneys, vesiculourateral reflux,,11 ectopic right ureteric orifice, left hydronephrosis, left reflux and hyderoureter. In this case, we reported 8 rare urinary disorders co-exist in one patient, in which bilateral giant kidneys, urinary extravasation of right renal, bilateral megalo-ureters, left ureteral terminal obstruction, bilateral renal cyst and bladder emptying disorder have not been reported before.As one of the few treatable infertility, early diagnosis and treatment of KS are particularly important. Renal agenesis is the most common complication for KS patients, which may provide clues for the early diagnosis and treatment of the patient. Fourteen types of urinary disorders in the KS patients have been reported before, including renal agenesis ,KAL1 is a well-known gene most closely related to KS-associated renal agenesis. Moreover, renal agenesis was only found in male patients and occurred in 31.8% KS patients due to KAL1 defects, consistent with the percentage of renal agenesis (31%) in X-linked KS patients in the study by Quinton et al. Therefore, we could imply that renal agenesis may be a marker of KAL1 mutation in KS patients with an estimated sensitivity of 30%. In the present study, our patient was first detected with two-point mutations in the KAL1 gene. However, one is a G-to-A transition at position 1600 in exon 11, changing codon 534 from valine to isoleucine with a carrier rate of 59.16%, suggesting that mutation at this position has no genotype-phenotype correlation. The other mutation is a synonymous mutation of a C-to-T transition in exon 12. Next, we performed a whole-exome sequencing for the patient and identified a G-to-A transition in exon 2 in the PROKR2 gene, changing codon 178 from tryptophan to serine with a carrier frequency of 0.01813%. PROKR2 is located on the autosomal chromosome region 20p13 and is mainly expressed in the brain, olfactory bulbs, and testes. To the best of our knowledge, KS patients with a PROKR2 mutation presenting urinary disorders have not been reported elsewhere, and the associated genetic links were unclear. We suspected that a single mutation of KS-related genes may not have significant clinical performance, but multi-mutations may have an accumulation effect. Therefore, when co-mutation with other KS-related genes such as PROKR2, KAL1 with p.V534I mutation may lead to a novel presentation of urinary disorders. We also searched for KS patients with urinary disorders in the absence of KAL1 mutation. Remarkably, another KAL1 mutation at position c.1600G > A (p. V534I) in exon11 in a patient with bilateral abnormal rotation and bimanual synkinesis was found. Whether this mutation interacts with other KS-related genes to cause certain clinical phenotypes in KS, or other genes associated with KS-related urinary disorders need to be explored is yet unclear. More cases are required for genome, transcriptome, proteomics, and metabolomics analysis.In this report, the patient accepted bilateral testicular traction and fixation, and received HCG injection after the operation. As shown in Table 4KAL1 and PROKR2 in a case of KS for the first time, which enrich the integrity of urinary disorder types in patients with KS. Urinary disorders and cryptorchidism can be a hint for the occurrence of hypogonadotropic hypogonadism requiring the attention of doctors for early diagnosis and treatment. More studies on the genotype-phenotype correlation need to be done, especially between KAL1 mutation and KS related urinary disorders. Identification of this correlation may be helpful for an early and precise diagnosis, and lays the foundation of timely and effective treatment.In conclusion, we report 8 novel urinary disorders with missense mutations of 5Institutional review board/ethics committee approval was obtained from the Institutional Review Board of the First Hospital of Jilin University.We want to acknowledge the patient and his family for their support and cooperation.Conceptualization: Huining Tian, Xiaokun Gang, Guixia WangData curation: Huining TianFormal analysis: Huining TianFunding acquisition: Guixia Wang.Investigation: You Lv, Lin Sun.Methodology: Zi Yan.Project administration: Guixia Wang, Xiaokun Gang.Resources: Guixia Wang.Validation: Guixia Wang, Xiaokun Gang.Visualization: Guixia Wang, Xiaokun Gang.Writing – original draft: Huining Tian.Writing – review & editing: Zi Yan.


Similar to what has already occurred in cancer medicine, the management of cardiovascular conditions will likely be improved by non-invasive molecular imaging technologies that can provide earlier or more accurate diagnosis. These techniques are already having a positive impact in pre-clinical research by providing insight into pathophysiology or efficacy of new therapies. Contrast enhanced ultrasound (CEU) molecular imaging is a technique that relies on the ultrasound detection of targeted microbubble contrast agents to examine molecular or cellular events that occur at the blood pool-endothelial interface. CEU molecular imaging techniques have been developed that are able to provide unique information on atherosclerosis, ischemia reperfusion injury, angiogenesis, vascular inflammation, and thrombus formation. Accordingly, CEU has the potential to be used in a wide variety of circumstances to detect disease early or at the bedside, and to guide appropriate therapy based on vascular phenotype. This review will describe the physical basis for CEU molecular imaging, and the specific disease processes for the pre-clinical translational research experience. Non-invasive in vivo molecular imaging techniques have been developed to assess molecular or cellular phenotype in animal models of disease and in humans. These methods provide both temporal and spatial assessment of disease-related molecules, and are particularly impactful when they are combined with conventional methods for imaging function, structure and flow. There are many reasons from both the research and clinical perspective for the development of molecular imaging technology Fig.  1–3].In–3.In1–3]There are many different approaches to molecular imaging . One of Contrast-enhanced ultrasound (CEU) molecular imaging relies on the selective targeting and detection of encapsulated microbubbles (MBs) or other acoustically active micro or nanoparticles which are retained in tissue on the basis of their ability to bind to molecules. CEU has several unique advantages compared to other forms of molecular imaging. CEU images are able to be obtained very rapidly, within minutes of intravenous injection; and the lack of any need for extensive post-processing provides almost immediate information to the clinician. When compared to other technologies, CEU is balanced with regards to sensitivity and spatial resolution, which are often divergent when comparing modalities. CEU is also portable, inexpensive, and does not involve radiation; all of which are practical considerations for the use of the technology for population screening or even point-of-care assessment of patients. However, CEU is limited in terms of its molecular targets since conventional platforms for CEU using microbubbles can provide information only on events within the vascular compartment. In this review, we will focus on some of the basic principles for CEU molecular imaging and highlight most pertinent clinical applications where CEU molecular imaging may have a major impact.Conventional ultrasound contrast or enhancing agents are composed of MBs which contain a high molecular weight inert gas such as perfluorocarbons or sulfur hexafluoride, and have a shell composed of lipids, protein , or biocompatible polymers. The size of MBs that are typically used for cardiovascular applications in humans is generally in the range of 0.5−5 μm in diameter, which permits their intravenous injection and unimpeded passage through the microcirculation of the pulmonary, coronary and peripheral microcirculations , 8. The CEU molecular imaging is commonly performed using targeted MBs that have been retained by adhesion to specific molecules or cells that are within the vascular compartment. Targeting of MBs agents has been accomplished by two general approaches Fig.  . A simpA more specific approach for targeting of MBs is to conjugate disease-targeting ligands  to the surface of MBs. Targeting ligands can be ligated to the MB surface using any number of chemical conjugation strategies. The density of targeting ligands can reach several thousand per square micrometer of surface area. The performance of any MB bearing a targeting ligand is determined by many factors Fig. . Some ofThere are many methods that have been developed to specifically detect the non-linear signal that arises from stable cavitation of MB contrast agents in the blood pool . StudiesThe pathophysiology of atherosclerosis is complex and changes over the decades of disease development. However, a process common in both early disease development and late complications involves vascular inflammation . The abiFor the detection of aggressive atherogenesis, CEU molecular imaging of adhesion molecules such as selectins, intercellular-adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) has been applied in murine models of disease –23. ThesIn patients presenting with Acute Coronary Syndrome (ACS), the clinical diagnosis generally relies on history information, serologic markers such as troponins, and electrocardiogram (ECG). It is widely recognized that many patients do not have classic angina symptoms, and many of those with ACS do not have diagnostic changes on the ECG . In thosCEU molecular imaging has been developed to assessing ongoing or recent, but resolved, myocardial ischemia at the patient bedside. For such “ischemic memory” molecular imaging, MBs have been targeted to activated endothelium by either ligands for the endothelial selectin family of adhesion molecules which are expressed rapidly after ischemia, or by including phosphatidylserine (PS) into the shell of MBs Fig.  . TargetThrombosis has a key role in many pathophysiologic processes in cardiovascular disease. There are many different anti-thrombotic and antiplatelet therapies designed to inhibit thrombus formation or to accelerate dissolution of clot . BecauseAs mentioned above, prothrombotic processes involved in atherogenesis or complications of atherosclerosis have been imaged with CEU molecular imaging of VWF, platelets, fibrin and tissue factor. Targeted imaging using similar agents has also been applied to detect large thrombi within vessels or the cardiac chamber, or microvascular thrombosis such as that occurs with post-MI no-reflow phenomenon. The relative role each for each of these individual agents is influence by whether the thrombotic process of interest is fibrin-rich or platelet-rich; and the shear forces in the chamber/vessel involved. Fibrin-targeted agents have been shown to produce enhancement not only in areas of vascular mechanical injury, but also on ventricular thrombi in large animal models , 38. AgeWhether using molecular imaging for clinical diagnosis or for research purposes, the selection of the most appropriate imaging technique requires a consideration of the advantages and disadvantages for each approach. Because CEU unlike other forms of molecular imaging uses pure intravascular contrast agents, the most common cardiovascular disease-related processes imaged include endothelial activation, leukocyte recruitment, angiogenesis, and thrombus formation. In general, MRI and CT are considered to have the highest spatial resolution, while radionuclide imaging  tends to have the highest sensitivity and dynamic range for detecting tracer. CEU molecular imaging provides a good balance between spatial resolution and sensitivity. CEU also has the advantage of being able to provide near simultaneous structural and molecular imaging. With regards to temporal resolution and speed of acquisition, CEU can be performed with rapid acquisition times of less than 10 min, while commonly used PET/SPECT and MR protocols require anywhere from just under an hour to a day between contrast injection and imaging. Ultrasound is the only technique that can be used for bedside diagnosis and is the lowest cost for all advanced imaging modalities.With all of the considerations above in mind, in the clinical setting, CEU molecular imaging is an ideal approach when rapid point-of-care decisions need to be made, such as ischemic memory, or for use as a screening technique in large populations, such as in atherosclerotic risk assessment. However, the translation of these technologies to humans continues to be challenging. One major obstacle to clinical adoption is the complex nature of regulatory agencies that oversee the Good Manufacturing Processes (GMP) including Chemistry, Manufacturing and Control (CMC) validations. Another major hurdle has been the difficulties in funding large trials that will be needed to show not only efficacy, but changes in outcome based on incremental value in large clinical trials. Recently, a microbubble agent targeted to Vascular Endothelial Growth Factor (VEGF) receptors has been translated to humans for potential use in cancer detection. However, these trials were small exploratory trials without controls , 44, and


Antimicrobial resistance (AMR) is an emerging global health concern, affecting both the animal and the human population. Transmission pathways of AMR are therefore abundant and complex, and ways to prevent or reduce transmission to consumers must be identified. The overall goal of this study was to define the content of an intervention study aimed at reducing the transmission of AMR from animal sources to humans. To identify the most relevant pathways, Swiss experts and consumers were interviewed about their opinions on the risks of transmission of AMR. Opinions of experts and consumers were then qualitatively compared and the main gaps identified. The results revealed that Swiss consumers had several misconceptions regarding the sources and transmission of AMR, and that they in particular underestimated the importance of poultry meat and pets as a potential source of AMR. Furthermore, high uncertainty was noted in experts regarding the prevalence of AMR in pets and the potential of transmission to their owners. Consequently, awareness of AMR transmission pathways should be increased among consumers to overcome common misconceptions, which will help reduce the risk of transmission. Further research is needed to better understand the pets' potential to harbor and transmit AMR to their owners, and to identify most effective methods to increase risk awareness in consumers as well as intervention strategies promoting consumer behaviors to mitigate AMR transmissions at the human-animal interface. In recent decades, antimicrobial resistance (AMR) has become a major global threat to public health, resulting in higher mortality, and prolonged illness in both human and animal populations . The acqvice versa have been described. Another potential source of AMR for humans is mishandled raw meat, which can be contaminated with AMR determinants due to cross-contamination either during the slaughtering of the animal or later during the processing of the meat. If contaminated raw meat is handled with insufficient hygiene measures, for example during meal preparation in the kitchen, AMR genes can be transferred to the consumer . The second step investigated Swiss consumer perceptions and behavior regarding AMR and AMR transmission pathways around foods and pets (Study 2). In the third step, the identified AMR sources and pathways (outcome of Study 1) were compared to the perceptions and behaviors of consumers (Study 2). In this way, differences between the expert assessments of the importance of exposure pathways and the perceived importance of these pathways among consumers were identified. The identification of such gaps will foster the development of contents for targeted intervention studies for consumers.A literature search was conducted to collect quantitative information on the prevalence of AMR hazards originating from different animal sources and evidence for their transmission to humans. Because of the very broad research field (no selection of a specific AMR determinant and consideration of many different source compartments), the relevant literature reviews were screened in a preliminary step and the relevant quantitative data was extracted. Whenever available, Swiss data was collected. If Swiss data was lacking, international research data was considered. For the purpose of this investigation, only transmission pathways from animal sources to humans were considered and not vice versa. The pathways assessed included direct transmission via animal–human contact and indirect contact through the exposure to food of animal origin. Also, we included fresh produce as food of non-animal origin since a contamination from animal sources is likely . Moreover, the inclusion of fresh produce allowed the coverage of a wider spectrum of foods of relevance to consumers. Milk was excluded from the assessment, since raw milk is a negligible source of AMR hazards on the Swiss market. The main relevant AMR sources and transmission pathways for Swiss consumers and specific subpopulations are shown in www.nrp72.ch) of the Swiss National Science Foundation (SNF) were selected and invited to the workshop. Of these, seven participated in the workshop. The experts' main fields of expertise regarding AMR included small animal medicine (n = 1), ruminant medicine (n = 1), environmental sciences (n = 1), food safety sciences (n = 2), human medicine (1) and veterinary public health (n = 1). Two additional experts could not attend the workshop and were interviewed in person or through an online phone call prior to the workshop .Following the literature review, an expert workshop was organized to assess the relative relevance of selected AMR transmission pathways (September 2018). Thirteen researchers involved in projects funded as part of the National Research Program “Antimicrobial Resistance”  applicable to AMR hazards originating from animal sources . It was Our study did not focus on any specific AMR microorganisms or genes; rather, it aimed at considering the overall abundance of AMR as a hazard. The hazard was thus defined as an “AMR hazard,” which comprised both microorganisms and determinants to which resistance is expressed. AMR in non-pathogenic bacteria was also considered, whereas intrinsic AMR was excluded.The release assessment considered the abundance of AMR hazards originating from a specific source and thus considered the prevalence of different resistant bacteria or AMR determinants from identified sources.The exposure assessment considered both the recipients' intensity of contact with a specific source and the likelihood of AMR transmission. It thus assessed the likelihood of transmission of AMR hazards from a specified source to humans depending on the intensity of contact with the specific source, given that an AMR hazard is present. The exposure assessment did not consider the frequency of exposure to a specific source, but it focused on the likelihood of transmission during a contact.A summary of the most relevant data published in regard to the prevalence of AMR hazards and their transmission from different sources to humans in a Swiss context was presented to the experts as a basis for discussion among them. The release assessment was then performed interactively during the workshop. Using visualization material, a vertical scale representing the prevalence of AMR hazards was displayed on a pin board, labeled from “very low” to “very high.” In a step-by-step procedure, the different sources were placed on the scale considering the evidence from the literature and modifications from the plenary discussion. Discussion was stopped when a joint agreement of the experts regarding the relative positioning of sources was reached. The focus of the position of different sources was put on the relative position between different sources rather than the absolute position on the scale. The same procedure was then repeated for the exposure assessment.To estimate the frequency of exposure to AMR hazards for the population, person days at risk were calculated for relevant subpopulations in Switzerland. Person days at risk were calculated by multiplying the number of exposed people in the subpopulations by the number of days exposed to the respective source per year. The number of exposed people was mainly derived from Swiss national statics data, and the number of exposure days per year were estimated by the authors and verified in the framework of the expert workshop. All formulas, references and calculations for the generation of person days at risk are provided in the Staphylococcus aureus (MRSA).During the expert workshop, the experts suggested to limit the assessment to AMR hazards that are clinically relevant for humans in order to facilitate the ranking process. However, the present study aimed to avoid this limitation to AMR hazards where only single relevant bacteria or resistance determinant is taken into account. Special emphasis was thus placed on consideration of the comprehensive scope of AMR. Despite all efforts toward an unbiased assessment, it was recognized that the expert judgement was still likely to be biased toward clinically important AMR hazards, as most of the experts' daily work focused on clinically relevant AMR hazards such as extended-spectrum beta lactamase (ESBL) producers or methicillin-resistant We also acknowledge that only a limited number of experts contributed to the study, however, all relevant disciplines were covered by at least one expert. A number of experts worked in multidisciplinary environments and were thus able to provide inputs for several compartments. It also needs to be considered that we did not aim to capture individual opinions, but rather stimulate the discussion within the group and toward finding the best agreement.Considering the final output , releasewww.sichergeniessen.ch). For fresh produce, both the release and the exposure were considered low, whereas the person days at risk for this source were highest.The most distinct result was obtained for poultry meat, which ranked the highest by far according to the experts in terms of release. This was not surprising, and uncertainty among experts was therefore low. In contrast to the release, the uncertainty expressed by experts was higher in terms of exposure through poultry meat, which was also the case for other raw meat sources. Raw poultry meat ranked lower in the end than other meat sources on the exposure scale, because it was expected that consumer awareness regarding hygienic measure was higher for poultry meat than for other type of foods, and most likely lowest for fresh produce. One reason for the assumed high public awareness may be various nation-wide food safety campaigns launched in the past  “Richtig zubereiten—sicher geniessen” [“Proper preparation—safe enjoyment”], Farmers ranked highest in terms of human exposure, which is understood as the probability of transmission of AMR hazards from the source. Arguments to justify this high position were the close and sustained contact between livestock and farmers and the additional burden through substantial environmental contamination, such as the exposure through dust. Following this argument, veterinarians were ranked much lower in terms of exposure, because the application of more stringent sanitary measures was assumed.Although pets ranked low as a source of AMR release, the exposure of pet owners was assessed as high because of the presumed close contact between pets and their owners. However, the experts' uncertainty regarding the position of the pets in the overall framework was rather high. Furthermore, experts were at odds regarding the number of days at risk for pet owners. There was a debate regarding the extent to which a pet could actually pose a risk as an AMR reservoir to its owner while not being subject to antimicrobial treatment. Only a few studies have investigated the existence of AMR hazards in healthy dog populations, however most of them focused on a specific AMR hazard e.g., , 45, 46], 4645, 4Although not being part of the final framework, the experts discussed the burden of the environmental compartment. Again, the uncertainty was very high in terms of both release and exposure. While a number of publications address AMR in the context of farmland and food production, literature on AMR determinants in natural environments seems to be very limited. Even if there was substantial release from the environmental compartments such as soil or surface water, it was assumed that exposure during recreational activities such as hiking or biking would be low. The only relevant exposure mentioned was the direct intake of surface water, which might pose a certain risk for swimmers or scuba divers.A general limitation inherent to our assessment was that we disregarded simultaneous exposure to multiple pathways, and thus the potential accumulation of exposure to AMR sources (for example owning a pet and handling raw meat). Furthermore, this study only considered the release and exposure assessments of the different pathways, but not their consequences on the human side. The assessment thus does not take into account the clinical relevance for the exposed individual. Therefore, validity is only given for the specific purpose of this assessment, which was the overall exposure of humans to AMR hazards from different animal and food sources.The objective of Study 2 was to uncover the potential perceptions that consumers  have about AMR in general and about the possible exposure pathways. Furthermore, the study aimed to assess consumers' food- and pet-related behaviors in terms of hygiene.Participants were invited for an interview on safe food handling or safe pet handling. They were recruited through online portals and advertisements in supermarkets in the German-speaking part of Switzerland. The inclusion criteria for interview participation were a high level of spoken German, a minimum age of 18 years and either food preparation at home at least two times a week or the ownership of a pet (preferably cat or dog). A limitation is therefore, that the current study was focused on consumers from a specific part of Switzerland only. Each participant was interviewed on both transmission pathways of food and pets. Although the sample size was small, care was taken that the participants covered various characteristics of the consumer population . Seven males and seven females participated in the interviews, ages ranging from 23 to 63 years. Recruiting and interviewing continued until saturation had emerged after the fourteenth participant, i.e., when no new information was obtained from the interviews. Saturation is a common and established stopping rule in qualitative interview studies . AlthougThe interviews took place at the participants' homes between June and August 2018, lasted from 45 to 60 min and were carried out in German. All interviews were conducted by the same person. Participants were only informed about the broad topic of the interview, i.e., “safe food handling” or “safe pet handling.” The interviewer assured anonymity and confidential treatment of the data and obtained approval to tape the interview. The interview was only conducted after the participant had provided written informed consent. Interviews followed a guideline. At the end of the interview, the purpose of the study was explained in detail to the participants and they could ask questions. They then received 50 CHF as compensation.Based on a literature review and team discussions, two interview guidelines were developed: one started with the topic food preparation and one with the topic pet care . The intInterviews were transcribed using the F4 transcription software , and coded and analyzed using the MAXQDA software . The codes were developed based on the interview guidelines. Additional codes were identified while reading the interviews. All interviews were coded by the same researcher. Additionally, six interviews were coded by a second coder and then compared with the first coder. In case of conflict, the two coders discussed their findings and sought a compromise.Overall, interviewees were not aware that AMR can be contracted through food products or close contact with pets. Food preparers perceived genetically modified foods, food additives  and the use of agricultural pesticides  as the main health hazards related to food products. Food poisoning and stomach problems were also quoted but without noting bacteria, let alone AMR. Pet owner concerns referred to overtaxing, poor quality pet training, injuries from bites, and scratches and allergies. Only a few interviewees mentioned the possibility of pathogen transmission from pets to humans such as tick-borne diseases or infections with tapeworms, but did not specifically refer to AMR bacteria. The increased awareness of these hazards might result from the fact that they are more frequently addressed by veterinarians.I don't know, if an illness can be an issue. I didn't hear about it often. I mean an infection from animals to humans. A few things exist, but I don't think it is a big danger.” [Assessment of hazards through pets] .The majority of participants were familiar with the existence of AMR, the emergence of AMR in humans through the use of AM and possible negative consequences such as an increased risk of a longer course of disease.Well, antibiotics are effective against bacteria. It means antibiotics won't be effective anymore. It can't be used any longer for an illness usually treatable with it. In worst case scenario, it can be life-threatening. [Meaning of AMR] Furthermore, some participants mentioned that risks of AMR are particularly high for those with a compromised or incompletely developed immune system, such as the young, elderly, pregnant, and those with immune function-reducing conditions.Substantial knowledge gaps emerged regarding the transmission pathways of AMR. Interviewees did not feel well-informed about the topic. They believed that only the use of AM results in resistance, without being aware of the fact that AMR can spread between different sources. Public places , places in nature  and the exchange of body fluids  were mentioned as known transmission routes. Contact through feces, skin, hair, salvia, blood, or other fluids were reported as possible exposure routes for AMR between animals and humans. Respondents did not distinguish between the various types of infectious agents .We also identified some confusion regarding the term bacteria. First, most participants did not differentiate between the transmission of AMR bacteria and bacteria not resistant to antibiotics. Their one-dimensional concept of bacteria included health-preserving bacteria, harmful bacteria and AMR bacteria without differentiating between their various sources and consequences. Some participants emphasized the important role of bacteria in our bodies. At the same time, the source of AMR bacteria was frequently associated with dirt.I think bacteria are on dirty places and a lot of dirt is outside. [Assessment of transmission pathways] Second, most participants assumed that carrying AMR bacteria would always cause physical symptoms and thus be noticed. Third, a number of participants believed that AM are effective against bacteria and viruses, which consequently leads to a misconception with regard to the biological mechanisms underlying the spread of AMR.Participants' understanding of the risks of AMR was mostly based on their personal experiences. Very few of the interviewees had previous experience with AMR bacteria causing a life-threatening infection among family members, friends or colleagues, so they perceived little risk in AMR. In line with the optimism bias , 48, theEverything has to be declared in Switzerland, and we have the choice what we want to eat. Thus, the risk is relatively low here. It is quite safe to buy food in Switzerland. My biggest concern abroad is to get a bacterial infection. [Assessment of food products with an AMR risk] Most participants viewed animal products, especially meat, as more susceptible to AMR than products of non-animal origin. Some explained this by the use of AM in livestock farming. Others believed this because they either knew these foods belong in the fridge or that protein foods from animals constitute a high risk for food poisoning.Salmonella. Campylobacter was not mentioned, although it is the most common food-related zoonotic agent causing diarrhea in Switzerland The fact that interviewees rated traveling abroad as more risky than food products or pets can be explained by people's unfamiliarity with foreign regions. Risk perception is related to factors such as newness, controllability or observability, whereby unfamiliar and dreadful hazards are perceived as most risky . Furtherclean, separate, cook and chill, as recommended by the FSVO .chill, some interviewees knew that a certain temperature for refrigerators is recommended without knowing the exact degrees. They were not aware that refrigeration slows down bacterial growth. Spoilage of food was associated more strongly with mold than bacteria.Regarding cook measures, participants were aware of concerns regarding undercooked meat because “something” could happen, but not everybody knew that the goal of heating food to a high temperature is to destroy active bacteria. Nobody reported validating the accuracy of the meat's temperature with a thermometer.Concerning Later in the interview, food preparers were asked about measures to prevent AMR transmission in the kitchen . Most ofAvoiding close contact with other animals. Or even with humans, but I don't want this. Paying attention to what my dog eats outside. I could wash him, or maybe go to the veterinarian regularly. [Measures to avoid AMR on pets] Surprisingly, some pet owners did not know whether medicine prescribed for their animal in the past was antibiotics or something else. Probably as a result of their low problem awareness, they had not asked the veterinarian about the prescribed treatment.A disparity was apparent in our sample between participants' knowledge of possible hygiene measures and self-reported practices . The awaAll participants mentioned a lack of knowledge about AMR and preventive measures as the main barrier preventing them from carrying out food or pet safety actions. Since knowledge gaps about AM and AMR have been found to be a critical determinant of non-adherence to physicians' instructions , they miOur data also indicated there was a low sense of personal ability to help to contain the problem because infections were perceived as outside the influence of individuals. People who perceive that conditions are due to forces beyond their control tend to take more risks , which mI can't control whether my cat comes back home with AMR or not. What can I do about it? [Measures to avoid AMR on pets] .Even if some respondents recognized AMR as a problem in the community, most did not feel they had a personal role in either the problem or the solution. Interviewees believed that improving and maintaining food safety should be achieved mainly by farmers and food producers higher in the food supply chain and prior to food offered for sale, which is a denial of responsibility . The facThree important differences regarding risk estimates of meat products emerged between experts and consumers. First, experts clearly distinguished between raw and processed meat in their risk estimates as the risk of contaminated meat is reduced by cooking or other modern sanitary techniques to kill pathogenic germs . Thereforelease of AMR hazards much higher for raw poultry than for raw red meat , raw fish or raw seafood, while they rated the exposure risk as the same for these food products. Experts namely expected that lay people's awareness regarding hygienic measures is higher for raw poultry meat than for other types of raw meat. However, most lay people underestimated the risk of raw poultry meat compared to other raw animal food products. Thus, they do not implement preventive measures as expected or recommended by experts (Second, experts rated the  experts . GeneralThird, lay people believed that organic and Swiss-produced meat is not affected or is less affected by AMR, a common misconception in consumers . They shrelease from healthy pets to be high. The exposure for pet owners was assessed as rather high, because of the presumed close physical contact of pets and their owners. The interview study confirmed that pet owners were in close contact with their pets but interviewees' risk perception regarding the AMR release by their own pets was extremely low. Contrary to transmission pathways at the human–animal interface, lay people generally perceived the AMR risk through the environment to have a greater impact than animal sources. In comparison, experts assessed the risk of AMR contraction for recreationally active people due to the release of AMR from the environment or surface water to be very low.Regarding pets as an exposure source, experts acknowledged research showing that animals may act as reservoirs for resistant organisms, especially during acute infection or AM treatment. However, as mentioned above, experts regarded the uncertainty about the AMR The gaps between experts' and consumers' assessments might in part have emerged due to the different assessment criteria used to rank various AMR transmission pathways . AlthougThe ultimate aim of this paper was to identify the relevant content and direction for an intervention aimed at consumers to reduce their exposure to AMR hazards from animals and food sources. Although the present results are linked to the Swiss context, they may be transferred to other regions that have a comparable AMR situation, consumer culture and food safety standards. Taking into consideration the specific conditions such as prevalence of AMR in different sources, relevant subpopulations and food and pet handling habits of the target population, the assessment can also be applied in a different geographical and cultural context.In the present context, practical implications could be gained from the comparison between expert opinions and lay peoples' perceptions of different AMR exposure pathways. Interventions aimed at food preparers should address the potential risk of AMR hazards in raw meat. Specifically, raw poultry meat should be emphasized as risky because of its high AMR release, and because consumers underestimate the increased risk of pathogens in poultry meat. Additionally, it is important to communicate that organic and local meat products are also potential AMR sources [e.g., Garcia and Teixeira , althougWe also recommend educating lay people about these facts in order to raise their awareness about the spread of AMR . Providiclean, separate, cook and chill” recommendations for food preparation. In addition, quantitative studies are needed to indicate the psychosocial determinants underlying pet owners' preventive behavior. Interventions can then be targeted toward these determinants in order to reduce pet owner exposure to potentially harmful AMR or other microbiological hazards originating from their pets.To date, quantitative data about the prevalence of AMR in pets and the likelihood of transmission to their owners or veterinarians is scarce. However, taking into account the large number of pet owners in Switzerland (3.5 million; see To conclude, awareness about AMR transmission pathways through food and animals should be increased among lay people and common misconceptions be overcome. Additionally, further investigations are needed to quantify the release of AMR in pets and the likelihood of transmission to their owners.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.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 for participation was not required for this study in accordance with the national legislation and the institutional requirements.IL and CF designed, collected and analyzed the data, and drafted the manuscript. VV and KS supervised the project work, contributed to the concept and design of the study, and revised the manuscript. VV acquired the funds. All authors contributed to the article and approved the submitted version.IL and KS were employed by the company SAFOSO AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Chest pain is a common presentation, and there are a wide variety of ways in which it can be investigated and treated. There is growing interest in whether the way we reach a diagnosis of angina can affect the long-term prognosis. In addition to its unparalleled negative predictive value, computed tomography coronary angiography (CCTA) gives anatomical information on the extent and severity of coronary artery disease. This article discusses recent research into the ability of CCTA to predict and improve long-term prognosis for patients with stable angina.Results from retrospective studies, randomised controlled trials and meta-analyses all suggest that initial investigation with computed tomography coronary angiography confers a prognostic benefit. In addition, the most recent studies have shown that the assessment of plaque burden and plaque constituents is predictive of long-term outcomes.Management of stable chest pain should be guided by a CCTA-based approach. Future research should focus on whether incorporating plaque analysis strategies into clinical practice confers additional benefit. To improve upon prognosis, one must make an accurate diagnosis. The term prognosis was coined by Hippocrates, to mean “foreseeing and foretelling” . When doTo understand how CCTA can affect the long-term prognosis of patients with stable angina, we need to understand how it is managed. Chest pain is a common presenting complaint, contributing nearly 4% of all new consultations to general practitioners . A healtHistorically, stenoses deemed ‘significant’  would be treated with revascularisation—a treatment with proven prognostic benefit in acute myocardial infarction . Early sToday, there is a plethora of investigations available for patients with stable angina, and each technique has unique benefits. Thus, it can be difficult to decide which test is best as a first line, with even international guidelines presenting conflicting recommendations Table  4, 12–1–112–14. Multiple studies have established the excellent diagnostic accuracy of CCTA compared to invasive coronary angiography to identify coronary artery stenoses , 16. TheCCTA allows accurate quantification of plaque burden which has been shown to improve the ability to risk stratify patients when added to conventional angiographic assessment Fig.  21]. In. In21]. CCTA not only provides information on anatomy, but can also be used to gain functional information on the significance of coronary artery stenoses. CT-FFR calculated from CCTA using computational fluid dynamic techniques has been shown to improve the accuracy of identifying haemodynamically significant coronary artery disease . In the In addition to the burden of atherosclerotic plaque, particularly subtypes of plaque have been shown to confer a worse prognosis. Non-calcified plaque on CCTA is associated with a worse prognosis compared to calcified plaque , 30. VisIt is now also possible to perform quantitative assessment of atherosclerotic plaque subtypes on CCTA. In the Incidental Coronary EveNts Identified by Computed Tomography (ICONIC) registry, quantitative plaque burden, fibrofatty volume and necrotic core volume on CCTA were associated with risk of subsequent ACS . In the Accurately identifying the presence of coronary artery disease has the potential to alter diagnoses, and subsequently management, for patients with stable angina. Registry studies have established the ability of CCTA to reclassify patients compared to clinical risk scores in up to two thirds of patients •. In thep = 0.004). The improved prognosis was not driven by increased revascularisation, but rather, the impact of increased medical therapy, particularly in the group with non-obstructive coronary artery disease [The 5-year outcomes of the SCOT-HEART trial provided the first evidence that management based on CCTA findings could improve clinical outcomes ••. In th disease . These f disease . Amongst disease . Interes disease , 48.TablAn early criticism of CCTA was the potential to increase the use of invasive coronary angiography. In the PROMISE trial, rates of referral for invasive coronary angiography were higher in patients undergoing CCTA compared to functional imaging, but those undergoing CCTA were less likely to have normal coronary arteries . FurtherWhilst the ability of CCTA to reduce cardiovascular death and non-fatal myocardial infarction has been discussed, one of the CCTA’s greatest strengths is the ability to identify patients with normal coronary arteries. An interesting finding from the SCOT-HEART study was that the greatest quality of life improvements was seen in the cohort of patients who had normal coronary arteries and did not require lifelong medical therapy .Guidelines in the United Kingdom from the National Institute for health and Care Excellence (NICE) are currently the only ones that recommend CCTA as first-line assessment, doing away with pre-test probability calculations Table  4, 12, , 12, 14.Randomised trials and real-world data show CCTA that can be used to assess long-term prognosis in patients with stable angina. It provides the ability to target optimal medical therapy and prognostic preventative medications to those who will benefit from them. Moreover, invasive management can be directed to those with disease in high-risk locations. In addition, novel techniques can be used to qualitatively and quantitatively characterise high-risk plaques. Management based on CCTA has been shown to improve long-term outcomes and this is reflected in national and international guidelines.


Tmc1 (mTmc1ex1 and mTmc1ex2) were reported to be expressed in the cochlea of infant mice, though only the sequence of mTmc1ex2 had been deposited in GenBank. However, due to the presence of an upstream open reading frame (uORF) and the absence of a typical Kozak sequence in mTmc1ex2, we questioned whether mTMC1 was translated from mTmc1ex2. Therefore, in this study, we evaluated which splice variant was protein-coding mRNA. Firstly, the results of RT-PCR and cDNA cloning of mTmc1 using mRNA isolated from the cochlea of five-week-old mice suggested that more Tmc1ex1 were expressed than mTmc1ex2. Secondly, mTMC1 was translated from mTmc1ex1 but not from mTmc1ex2 in a heterologous expression system. Finally, analyses using site-directed mutagenesis revealed that the uORF and the weak Kozak sequence in mTmc1ex2 prevented the translation of mTMC1 from mTmc1ex2. These results suggest that mTmc1ex1 plays a main role in the expression of mTMC1 in the mouse cochlea, and therefore, mTmc1ex1 should be the mRNA for mTMC1 hereafter.Mechanical stimuli caused by sound waves are detected by hair cells in the cochlea through the opening of mechanoelectrical transduction (MET) channels. Transmembrane channel-like protein 1 (TMC1) has been revealed to be the pore-forming component of the MET channel. The two splice variants for mouse  Tmc1 have been reported to cause hereditary deafness in mice and human [Transmembrane channel-like protein 1 (TMC1) forms the mechanoelectrical transduction (MET) channel, a type of mechanosensitive channel, at the tips of stereocilia of hair cells in the inner ear. When the stereocilia are tilted by sound wave or gravity, the MET channel opens and transduces these force into electrical signals by depolarizing membrane potentials [Tmc1 (mTmc1) gene, two splice variants (mTmc1ex1 and mTmc1ex2) were reported [mTmc1ex2 includes exon 2 containing its start codon, but mTmc1ex1 excludes the exon 2 and uses AUG (ATG in the case of complementary DNA (cDNA)) in exon 1 as its start codon [N-terminal amino acid residues of mTMC1ex1 and mTMC1ex2 are different (mTmc1ex1 and mTmc1ex2 were similar to each other in either utricular maculae or cochleae at least within the first two weeks after birth in mice [As transcripts of the mouse ifferent B. ExpresmTmc1 mRNA deposited in Genbank had been only mTmc1ex2 (accession number: NM_028953.2). .) However, we questioned whether the mTMC1 protein is translated from mTmc1ex2 for the following two reasons. first, the sequence around the start codon of mTmc1ex2 (tctTttATGT) does not match the typical Kozak sequence , which is the consensus sequence for translation initiation [mTmc1ex2. On the other hand, the sequence around the start codon of mTmc1ex1 (ttcAggATGC) contains a crucial A at the −3 position. Second, there is an upstream open reading frame (uORF) in exon 1, translation of which starts with ATG for mTmc1ex1  or the canning” ).mTmc1ex1 and mTmc1ex2 using a heterologous expression system. Our results suggest that mTmc1ex1 is the splice variant which functions as mRNA for mTMC1.Therefore, in this study, we examined which splice variant was mainly expressed in the cochlea of mature mice and whether mTMC1 was translated from mTmc1ex1 and mTmc1ex2 in the cochlea of five-week-old mice. By using primer pairs which can amplify either mTmc1ex1 or mTmc1ex2 specifically, both mTmc1ex1 and mTmc1ex2 were amplified . The size of the amplicon appeared to be less than 4000 bp, indicating that the amplicon was composed of mTmc1ex1 . With the primer pair, the band derived from mTmc1ex1 was mainly amplified (the shorter band) but the band from mTmc1ex2 (the longer band) was faint , which expresses both a protein encoded by the inserted cDNA and an enhanced green fluorescent protein (EGFP). By detecting EGFP expression, we can confirm the success of transfection. When the vector containing mTmc1ex1 was transfected into HEK293T cells, the mTMC1 protein was detected by western blotting (mTmc1ex1 full untranslated region (UTR) ). On the other hand, when the vector containing mTmc1ex2 was transfected, mTMC1 was not detected (mTmc1ex2 full UTR (WT) with no significant difference from a group transfected with an empty vector ). Similar results were obtained even when another expression vector was used (pcDNA3.1(+), mTmc1ex1 is a splice variant from which mTMC1 is translated and that mTmc1ex2 does not function as an mRNA for mTMC1.Next, we examined whether the mTMC1 protein can be translated from mTmc1ex2. First, a removal of uORF from 5′ UTR of mTmc1ex2 did not increase the expression of mTMC1  slightly increased the expression of mTMC1 (mTmc1ex2 (b). A combination of the removal of uORF and the addition of the typical Kozak sequence significantly increased the mTMC1 expression (mTmc1ex1 (vs. (a)). Similar results were obtained using pcDNA3.1(+) also  also . These rmTmc1 gene. Although the difference between mTMC1ex1 and mTMC1ex2 is only a few amino acid residues at their N-terminus  is equivalent to mTmc1ex1, and its N-terminal amino acid residues  resembles that of mTMC1ex1 (MPPKKVQI…) rather than that of mTMC1ex2 (MLQI…). Moreover, a splice variant of hTMC1 equivalent to mTmc1ex2 has not been reported as far as we know. Therefore, this information on hTMC1 also supports that mTmc1ex1 is the mRNA for mTMC1.Our results strongly suggest that mTmc1ex1 should be used as the mRNA of the mTmc1 gene. If a primer is designed from the sequence of exon 2, the primer detects only mTmc1ex2. Therefore, the RT-PCR results using the primer will not give correct information on the expression levels of mRNA which translates mTMC1. Moreover, even if primers for RT-PCR and probes for Northern blotting are designed from the common sequence of mTmc1ex1 and mTmc1ex2 (the sequence other than exon 2), they detect both mTmc1ex1 and mTmc1ex2. The expression of mTmc1 has been reported in cochlea, eye, testis, and others in many cases without discriminating mTmc1ex1 and mTmc1ex2 [mTmc1 mRNAs are mainly mTmc1ex2, the actual expression level of the mTMC1 protein will be low. Therefore, when mTmc1 mRNA is detected, it should be done in a mTmc1ex1-specific manner.One significance of our findings is that they show that mTmc1ex2 ,8. If thmTmc1ex2 [Moreover, although the difference in amino acid sequence between mTMC1ex1 and mTMC1ex2 is only a few amino acid residues, the mTMC1 protein which is composed of the genuine amino acid sequence  should be used for analyses in heterologous expression systems. In heterologous expression systems, in order to increase the expression level, the Kozak sequence is sometimes added to the sequence before the start codon, as done in a research on mTMC1 using mTmc1ex2 . Such anN-terminus between mTMC1ex1 and mTMC1ex2 might not cause any functional differences between them. At least, when they were heterologously expressed, their localizations  were not changed. However, it was recently reported that co-expression of KCNQ1, a K+ channel, rescued hTMC1 plasma membrane targeting [N-terminus of TMC1, the difference between mTMC1ex1 and mTMC1ex2 may affect their interaction with KCNQ1. Additionally, even when KCNQ1 was co-expressed, channel activities of hTMC1 were not detected, suggesting requirements of other components for the formation of a functional channel by TMC1 [The difference in a few amino acid residues at the argeting  althoughargeting . If the  by TMC1 . When thmTmc1ex2, it was reported that mTmc1ex2 was expressed at the level similar to mTmc1ex1 in the cochlea at least within the first two weeks after birth in mice [mTmc1ex2?Although our results suggest that mTMC1 is not translated from mTmc1ex2 might be expressed in order to reduce the expression level of mTMC1 protein in the immature cochlea. It was reported that, in the first week after birth, mTmc2 (an orthologue of mTmc1) was mainly expressed in the cochlea and mTmc1 expression was low [mTmc2 decreased but mTmc1 expression increased [mTmc1ex2 as a splice variant of mTmc1 in the immature cochlea may result in the lower expression of mTMC1 protein that might be useful for mTMC2 to play a major role in mechanoelectrical transduction instead of mTMC1. On the other hand, our present study indicates that mTmc1ex1 is the major splice variant of mTmc1 in the cochlea of 5-week-old mice. Therefore, the dominant expression of mTmc1ex1 may be advantageous for sufficient expression of mTMC1 in the mature cochlea.First,  was low . After tncreased  althoughmTmc1ex2 might have physiological functions. The uORF contains the Kozak sequence, which is identical to that of mTmc1ex1. Therefore, the peptide encoded by the uORF is supposed to be translated from mTmc1ex2 mRNA. The amino acid sequence of the peptide is MPPKKGVSGHF. As the peptide is very short and its first five amino acid residues out of 11 are identical to the N-terminus of mTMC1ex1, it will be difficult to specifically detect the short peptide in the cells which express mTMC1 using antibodies. However, there is a possibility that the short peptide has some functional roles as it has been reported that such short peptides encoded by uORF in some mRNAs were translated and showed physiological functions such as translational regulations [mTmc1ex2.Second, the peptide encoded by the uORF in ulations . FurthermTmc1ex1 plays a main role in the expression of mTMC1 protein in mouse cochlea, and therefore, mTmc1ex1 should be treated as the mRNA for mTMC1 hereafter. Furthermore, our findings recommend that researchers pay more attention to 5′ UTR sequences (uORF and Kozak sequence) in mRNAs of proteins in which they are interested in order to avoid conducting research on proteins which are not actually translated.In conclusion, the present study suggested that All animal experiments were performed in accordance with guidelines from and protocols approved by the Institutional Animal Care and Use Committee, Graduate School of Veterinary Medicine, Hokkaido University .mTmc1ex1 and mTmc1ex2, PCR was conducted using a high-fidelity polymerase , and the primers are listed in mTmc1ex2 cDNA (NM_028953.2). Simultaneously, whole mTmc1ex1 cDNA also can be amplified with these primers. The design of the other primers was explained in the Results section. The PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.RNAs were extracted from cochlea of male C57BL/6N mice  using NucleoSpin RNA II . cDNA was synthesized using PrimeScript II Reverse Transcriptase (Takara Bio). In order to evaluate the expression levels of mTmc1 cDNA was amplified by PCR using PrimeSTAR GXL and the primers “mTmc1 5′UTR F” and “mTmc1 3′UTR R” (shown in mTmc1 cDNA) was cloned in pGEM-T Easy Vector , and four clones were sequenced. The presence or absence of exon 2 in these clones were examined by PCR using a primer pair: “mTmc1 5′UTR F” and “mTmc1 R”. The mTmc1ex1 cDNA which contained no PCR errors was subcloned in a bicistronic expression vector, pIRES2-EGFP vector  or pcDNA3.1(+) vector (Takara Bio). In the case of mTmc1ex2, there was a deletion of 41 nucleotides in CDS in the only clone obtained , was artificially constructed. The mutants of mTmc1ex2 were made by site-directed mutagenesis using the PrimeSTAR Mutagenesis Basal kit (Takara Bio). Mutations were verified by sequencing.The full-length of shown in . The PCRd #20 in . Therefo2 incubator. Cells were transiently transfected with plasmids using TransIT-293 Transfection Reagent (Takara Bio). Thirty to forty-eight hours after the transfection, the cells were used for experiments.HEK 293T cells were obtained from the RIKEN BioResource Research Center through the National Bio-Resource Project of the MEXT, Japan. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and penicillin/streptomycin  at 37 °C in a 5% COg for 10 min at 4 °C. The supernatants were mixed with equal volume of urea sample buffer , and 0.1 M dithiothreitol (DTT) [Western blotting was conducted as described previously  with somol (DTT) ) and heamTmie-EGFP pcDNA3.1 (cloned from cDNA of cochlea) and mTmc1ex1 pcDNA3.1 or mTmc1ex2w/o uORF w/Kozak pcDNA3.1 on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. Nonspecific staining was blocked by incubations with image-iT FX signal enhancer (Thermo Fisher Scientific) and with PBS containing 2% bovine serum albumin and 5% normal goat serum. The primary antibody was a custom-made anti-mTMC1 polyclonal rabbit antibody against mTMC1 , Sigma-Aldrich). The secondary antibody was Alexa 555-conjugated goat anti-rabbit IgG F(ab’)2 fragment (Thermo Fisher Scientific). After washings, the coverslips were mounted with Prolong Diamond (Thermo Fisher Scientific). Fluorescence was observed using a confocal microscope .Cells which were co-transfected with


So, based on this, the role of the NLRP3-inflammasome during ageing and the inflamm-ageing is very evident. However, the complexity of the inflammasomes shows an independent role of these with respect to the canonical inflammatory pathways. Nlrp3 -/- mice have shown similar levels of other cytokines compared to their littermates controls. Youm et al., showed increased levels of IL-6 in serum from aged Nlrp3 -/- and WT mice [Inflammasomes are multiprotein complexes that are formed and activated after exposure to danger signals with the activation of caspase-1 causing the maturation and release of IL-1β and IL-18. In the formation of inflammasome complexes, several cytosolic pattern recognition receptors (PRRs) act as sensors of pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPS) . Many of WT mice . These fFurther investigation focused on the specific role of NLRP3-inflammasome in cardiac ageing will be required to address these issues and would open a new pharmacological target for the use of the specific NLRP3 inhibitor with respect to anti-inflammatory treatments. We invite to the scientific community to discuss this possible new topic.


The biofabrication of biomimetic scaffolds for tissue engineering applications is a field in continuous expansion. Of particular interest, nanofibrous scaffolds can mimic the mechanical and structural properties  of the natural extracellular matrix (ECM) and have shown high potential in tissue engineering and regenerative medicine. This review presents a general overview on nanofiber fabrication, with a specific focus on the design and application of electrospun nanofibrous scaffolds for vascular regeneration. The main nanofiber fabrication approaches, including self-assembly, thermally induced phase separation, and electrospinning are described. We also address nanofibrous scaffold design, including nanofiber structuring and surface functionalization, to improve scaffolds’ properties. Scaffolds for vascular regeneration with enhanced functional properties, given by providing cells with structural or bioactive cues, are discussed. Finally, current in vivo evaluation strategies of these nanofibrous scaffolds are introduced as the final step, before their potential application in clinical vascular tissue engineering can be further assessed.  Tissue engineering, which combines the principles and methods of the life sciences with those of engineering to create tissue replacements to direct tissue regeneration, has attracted many researchers with the hope of regenerating a patient’s own tissues and organs without the need for tissue/organ transplantation ,2. The mThe field of regenerative medicine has achieved significant progress in the past decade  4]. A f. A f4]. However, significant challenges remain before the widespread adoption of tissue engineering approaches in the clinic. Several important aspects should be considered for successful regeneration, such as (i) scaffold design with desired mechanical, chemical, and biological properties that better mimic a tissue’s native microenvironment and better support cell activity; (ii) maintenance and regulation of growth factors to steer cellular behavior; and (iii) vascularization, allowing integration of the engineered tissues into the host system for fully functional regeneration ,18. Tissue engineering approaches typically start with scaffold design. An engineered scaffold should provide favorable biochemical  and bioSelf-assembly is a method by which simple molecular and macromolecular building blocks are engineered to organize themselves through non-covalent forces, such as hydrogen bonding, hydrophobic forces, and electrostatic interactions ,42. The Supramolecular nanofibers can also be created by other self-assembling motifs. An early example of supramolecular polymers forming nanofibers is ureido-pyrimidone (Upy). Sijbesma and coworkers prepared the self-assembled UPy nanofibers through self-complementary four-hydrogen bonding between self-complimentary Upy moieties . The fibThe nanofibers produced by molecular self-assembly usually are much smaller than those produced by electrospinning, clearly due to the molecular design character of self-assembly . While fThermally induced phase separation (TIPS) has been used to produce three-dimensional (3D) porous scaffolds since the 1980s. Nanofibrous materials have also been made by this technique . When phL-lactic acid) (PLLA) nanofibrous matrices by using TIPS with a low polymer concentration from 1% to 7.5% in tetrahydrofuran . B. B91]. BThe shapes and morphologies of beads and fibers can be controlled by adjusting electrospinning parameters. For example, Lee et al. illustrated that changing polymer concentration during electrospinning could result in different bead dimensions and morphology. Bead structures were largely produced when electrospinning with a PS solution below 15%, while only fibers were formed after increasing the concentration above 15% . As PS iAligned nanofibers provide specific physical cues able to directly influence cell activity, especially by guiding cell distribution and elongation in the direction of the fibers . SeveralMoreover, aligned fibers also showed a positive effect on vascular regeneration. Zhu et al. fabricated PCL aligned fibers by using a rotating mandrel to collect fibers . Human uThe most common and effective way to fabricate aligned electrospun nanofibers is the use of a rotating collector B. A geneA honeycomb is composed of hexagonal wax cells constructed by honeybees in their nests . The natTo fabricate honeycomb patterns by electrospinning, two methods have proven successful: (1) self-assembly  and (2) In addition to honeycomb patterns, other studies reported achieving fiber patterns through directed fiber deposition, such as in the case of the direct writing approach ,121. LeeA conventional electrospun membrane only produces one type of (micro or nano) fiber. An improvement of recent electrospinning approaches is creating complex electrospun scaffolds with both micro and nanofibers at the same time. Electrospun fibers in the nano scale could mimic native ECM fibers (a diameter in the range of 10–50 nm), while microfibers create highly porous scaffolds which benefit cell infiltration and migration. The fabrication of electrospun scaffolds that contain spider-web-like nano-nets with two types of (micro or nano) fibers could enhance scaffolds properties for tissue engineering applications  and coulNylon-6 and poly (acrylic acid) nano-nets were first reported, which were fabricated by tuning electrospun parameters . Four yeDual air jet spinning (AJS) is another strategy with which to fabricate composite electrospun fibers with different diameters. The use of dual AJS could simultaneously control the mixing of two different polymers fibers from two AJS nozzles. This platform could afford the possibility of mixing two polymers together to produce web-like fibers with different diameters. Abdal-hay et al. reported that composite fibrous scaffolds composed of PCL and polyamide-6 (PA-6) fibers were successfully fabricated by using the dual AJS technique. Fiber diameter distribution showed that the diameters of PA-6 fibers were 278 ± 54 nm, while pure PCL had thick fibers with diameters of 2345 ± 152 nm. The composite fibrous scaffolds had better tensile properties than pure fibrous scaffolds. In vitro study demonstrated the PCL/PA-6 composite fibrous scaffolds provide a favorable microenvironment that supports attachment and proliferation of endothelial cells, so they could be promising scaffolds for vascular tissue engineering. Other studies have also shown spider-web-like structures in different electrospun polymeric membranes can better mimic the ECM and displayed enhanced mechanical properties, as in the cases here discussed. These scaffolds could be especially beneficial for cell attachment and migration in comparison to membranes without nano-webs ,128,129.D-threonine (Thr) and sodium hydroxide (NaOH) could self-assemble into helical nanofibers [2 nanofibers using that technique, with diameters of the fabricated nanofibers from 80 to 140 nm [4 flow in the carrier gas resulted in a helical structure of the formed nanofibers, the actual mechanism with which a helical structure was formed was not reported. The researchers hypothesized that CH4 helped with moderating the highly oxidizing environment and retained the activity of iron-catalyzed particles. Inspired by the structures of proteins in different tissues of our body, nanofibers with curved morphologies display many specific features, including large porosity, elasticity, and toughness, which make it attractive in biomedical tissue engineering. Three techniques has been reported to produce waved nanofibers: (1) self-assembly ,131, (2)nofibers . Peptideo 140 nm . AlthougElectrospinning has been regarded as the most common and efficient technique with which to fabricate helical nanofibers. Researchers produced helical nanofibers by electrospinning and studied the potential factors that could guide the fibers to be helical and curly. Zhou et al. created wavy nanofibers through an airflow-electrospinning process . Airflow2⋅HCl) could form nanotubes at physiological pH via self-assembly methods. To test the delivery of these cationic dipeptide nanotubes in cells, fluorescein labeled single stranded DNA (ssDNA) was bound to the nanotubes by electrostatic interactions. After incubation with HeLa cells, the fluorescence showed that these nanotubes could enter cells and accumulate in the cytoplasm. Nanotubes are also promising for potential applications of gene and drug delivery. However, low yield is a significant limitation to produce nanotubes in a large scale. The template-directed approach has also been used to build nanotubes, since it is more direct and controllable compared to the self-assembly approach [Tubular nanostructures are widely used in tissue engineering, especially for drug delivery . Many apapproach ,144. HouCompared with self-assembly and template-directed approaches, coaxial electrospinning was proved to be the optimal approach for creating continuous nanotubes, with controllable conditions and large-scale production. In co-electrospinning, a coaxial nozzle is the key device that is comprised of a central tube and a concentric circular corona. This coaxial nozzle allows two different solutions to produce a core-shell composite structure ,146. In Currently, nanofibers with different patterns have been successfully fabricated by adjusting the processing parameters of electrospinning. Nanofibers could be designed as specific structures depending on the biomedical applications, due to every structure having its own advantages. For example, aligned fibers were used to better mimic the collagen fibers in bone matrix . HoneycoTo explore more applications of nanofibrous scaffolds in tissue engineering, researchers investigated the functionalization of nanofibrous scaffolds by many different approaches, such as physical blends, core-shell electrospinning, and post functionalization . PhysicaUsing polymer blends is an easy and direct method to introduce bioactive components into nanofibrous scaffolds for tissue-engineering applications. Nanostructure polymer blends could result in the production of low-cost scaffolds with specific functionality. Biopolymers can be blended into a synthetic polymer for electrospinning to obtain better mechanical properties, good biodegradability, and good biocompatibility of nanofibers. For example, to improve the biodegradability and biocompatibility, gelatin was mixed into a PCL electrospun solution and then incorporated into PCL nanofibrous scaffolds during electrospinning . The obtThe combination of natural and synthetic polymers could effectively improve the mechanical properties and biocompatibility of nanofibrous scaffolds, which could support cell growth and favor tissue regeneration. More importantly, the simple physical blending process for the introduction of functional components to polymeric nanofibers usually resulted in converting the nanofibers into highly effective delivery systems. Many biofunctional components  . They fa2, O2, and NH3 have been used during plasma treatment to change the chemical composition or introduce functional groups on the surfaces of scaffolds for further applications. For example, polymer nanofibers were exposed with oxygen plasma in order to obtain carboxyl groups on the surface of nanofibers [2, –OH, and –COOH) on the surfaces of nanofibers, these surfaces could be largely used as substrates for biomolecule immobilization. Simple plasma treatment is a widely used method to improve nanofiber biocompatibility. Different gases such as Ar, Nnofibers . Plasma nofibers . After gA study demonstrated the functionalization of PLLA nanofibers with RGD (Arg–Gly–Asp) peptides by plasma treatments and covalent coupling. The plasma treatment of PLLA nanofibers is a key step to producing carboxyl groups for RGD coupling. RGD peptides could immobilize on the functionalized nanofibers by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) chemistry. In vitro results showed that RGD functionalized scaffolds promoted the proliferation and osteogenic differentiation of hMSCs . AnotherThe surface modification of nanofibrous scaffolds could also be achieved by wet chemical treatments. Wet chemical approaches, such as hydrolysis, and soaking in NaOH or KOH, often lead to the degradation of nanofiber properties and morphology. Chen et al. reported that PCL nanofibrous membranes treated with NaOH etching led to enhanced roughness and surface area . 3T3 fibClick chemistry is an attractive alternative for facile immobilization of bioactive components onto electrospun nanofiber surface, because of its simple reaction conditions, high reaction rate, high yields, and functional group tolerance ,192,193.The surface graft approach has been also developed to introduce functional groups on scaffolds for the subsequent coupling of bioactive components. Plasma treatment is usually required to introduce free radicals for polymerization during graft polymerization. For example, the surface of poly(vinylidene) fluoride nanofibers was successfully grafted with poly(methyl methacrylate) by plasma-induced graft copolymerization . By procVascular regeneration is one of the most challenging issues in tissue engineering ,206. DurNanotopography has been noted as an important factor with which to affect cell growth and differentiation. ECs could interact with their physical environments and be guided by a scaffold’s topography. Xu et al. reported that the alignment of nanofibers plays a positive role in the regulation of endothelial cellular behavior . Cell elApart from electrospun scaffolds, a few other studies showed that EC morphogenesis into capillary-like structures was regulated by micropatterned stripe substrates ,223. DikMany researchers have reported that the controlled release of angiogenic factors could promote vascular formation in vitro and in vivo. Electrospun nanofibrous scaffolds show potential to incorporate or functionalize bioactive components onto the scaffolds, and scaffolds designed for drug release have shown controlled delivery. VEGF is the most important growth factor to stimulate early vascular formation and promote angiogenesis. Jia and his colleagues loaded VEGF into the inner of core/shell fibrous scaffold by coaxial electrospinning with PLGA as the shell . VEGF reMany studies have also reported the immobilization of VEGF-mimetic peptides on scaffolds as a potential solution for vascular regeneration. D’Andrea et al. designed a VEGF-mimetic peptide, QK (domain: KLTWQELYQLKYKGI), and showed the ability to activate VEGF receptors and similar bioactivity to VEGF . QK pept2 to generate electrospun scaffolds with a functionalizable amine [Apart from VEGF-memetic peptides, RGD peptide was also reported to immobilize into electrospun scaffolds for vascular tissue engineering. For example, Kim et al electrospun a mixture of PLGA and PLGA-b-PEG-NHle amine . RGD was2S), a unique gasotransmitter that has been recognized as an important physiological and pathological signaling molecule, can mediate and promote the effects on angiogenesis [2S promotes EC proliferation and migration has been reported by different groups [2S has been reported to simulate angiogenesis in vitro and in vivo [2S has been recognized to be beneficial for angiogenesis, researchers started to focus on the development of H2S releasing scaffolds for vascular regeneration [2S donor, with PCL solution to form H2S release fibrous scaffolds [2S fibrous scaffolds could facilitate (H9c2 and 3T3) cell proliferation. Moreover, these scaffolds were also reported to increase the expression of collagen type I and collagen type III, and wound healing related genes. Kang and his colleagues synthesized a pH-controlled H2S donor (JK-1) from phenylphosphonothioic dichloride [2S releasing electrospun nanofibers [2S release. JK1 doped PCL fibers were nontoxic to fibroblasts and in vivo experiments proved that PCL-JK1 could significantly improve wound healing. Although previous studies showed promising results in vitro and in vivo, many aspects still need to be improved for the fabrication of H2S releasing nanofibrous scaffolds: (1) a slow H2S-releasing donor needs to be included; (2) in addition to physical doping, other surface functionalization approaches should be considered. Hydrogen sulfide  assay and rabbit or rat corneas are the most widely used animal models for studying the process of angiogenesis in vivo. CAM is an extraembryonic membrane mediating gas and nutrient exchanges until hatching . Since tin vitro. For a more thorough review on the several angiogenesis available assays, the reader is referred elsewhere [CAM assay has been used by many researchers as an in vivo model in vascular tissue engineering for more than 40 years. A number of studies have evaluated electrospun nanofibrous scaffolds on the CAM to examine their angiogenic response and biocompatibility. The general procedure of CAM assay is shown in lsewhere . Scaffolds for tissue engineering have become more and more important in regenerative medicine, which is crucial for the development of advanced clinical therapy for those critical tissue injuries that do not heal correctly by themselves . The key(1) Structure design.Most tissue regeneration, such as that of bone, muscle, and cartilage, necessitates 3D scaffolds, but nanofibrous scaffolds with high thickness are difficult to obtain by electrospinning. Electrospinning normally produces 2D random nanofibrous scaffolds with high porosity in the height of few microns . Honeyco(2) Nanofiber functionalizationTraditional nanofiber functionalization approaches lack of selective control. Ideally, functionalization could also be provided in specific locations in 3D scaffolds through the presence of selective reactive groups. For example, this could be reached by photopatterning and would allow for achieving spatial control of the fiber functionalization. The ability to use a photopatterning approach to create any spatial functionalization of fibrous scaffolds is a significant advantage, which could be used for the partial functionalization of bioactive molecules on fibrous scaffolds for vascular tissue regeneration. (3) Vascular tissue regenerationAn ideal scaffold should not only provide an appropriate physical environment ,253, whi