A significant number of new and advanced bioprinting technologies have been developed in recent years, enabling the generation of increasingly accurate models of human tissues both in the healthy and diseased state. Accordingly, this technology has generated a demand for smart bioinks that can enable the rapid and efficient generation of human bioprinted tissues that accurately recapitulate the properties of the same tissue found in vivo. Here, we define smart bioinks as those that provide controlled release of factors in response to stimuli or combine multiple materials to yield novel properties for the bioprinting of human tissues. This perspective piece reviews the existing literature and examines the potential for the incorporation of micro and nanotechnologies into bioinks to enhance their properties. It also discusses avenues for future work in this cutting-edge field.  Enginee3D bioprinting attempts to reproduce the three-dimensional organization of cells, replicating what the human body naturally does. This process can fabricate custom tissues or organs using patient-derived cells, thereby minimizing the risk of rejection after transplantation . The finCompared to non-biological 3D printing, 3D bioprinting requires additional levels of consideration, such as the choice of cell-adequate nutrient medium, cell type and growth and differentiation factors . Bioprin1–Rheological: Shear rate, shear stress, viscosity, and critical shear point of the bioink are the most significant rheologic parameters to assess if the bioink exhibits shear-thinning behaviour for improved print quality without impacting the bioink’s internal bonding structure . In othe2–Mechanical: The potential to regulate the mechanical characteristics of a bioink, such as elastic modulus, shear elastic modulus, Young’s modulus, or degradation rate . Control3–Biocompatibilitys: Biocompatibility is described as a substance’s ability to retain high cell viability, promote cell growth and proliferation, and preserve the cell population’s healthy, distinctive phenotype after exposure to a material .Making a hydrogel-based bioink requires that the desired cells are obtained in sufficient quantities and then added to the ink. The bioprinted structure must then be cultured in a medium enriched with nutrients designed to promote the appropriate cell growth and function. The selection of materials has a great impact on the biocompatibility, cellular viability, and mechanical behaviour of a bioprinted structure and thus care must be taken when determining the most suitable bioink for a given tissue engineering application. Most bioinks rely on crosslinking to turn the liquid bioink into a gel-like substance containing cells. Extrusion-based bioprinting often builds the desired structure through the addition of layers of cell-laden bioinks crosslinked to achieve the expected structures and their associated mechanical properties. Finally, these bioprinted constructs are cultured in media often followed by cellular and mechanical tests to characterize their constructs . AccordiThe use of functionalized bioinks in tissue engineering has resulted in a number of achievements for the healthcare industry, among which examples in the literature are reviewed in The development of 3D bio-printing bone and cartilage tissues also opened potential doors to new treatments for patients with arthritis, bone fractures, dental infections, and craniofacial defects. For instance, Mei et al. used a photo-cross-linkable hydrogel and additives, including nanoparticles (NPs), functional cells, and drugs/cytokine to make a bio-ink for bioprinting bone and cartilage . Such naOverall, these aforementioned studies demonstrate the broad range of functionalized bioinks of different compositions used for potential applications for 3D bioprinting. A subset of functionalized bioinks would include composite bioinks as seen in Various structures, including nanoparticles, nanofibers, microspheres, fillers and films, have been used for delivering drugs or therapeutic substances to tissues. These substances can promote cell growth, influence cell proliferation and differentiation, and control extracellular matrix (ECM) secretion . For insNanotechnology describes research applications focused on the principles and properties existing at the nanometric scale, at the level of atoms and molecules . The objGungor-Ozkerim et al. provide an in-depth discussion of the different bioinks currently employed for bioprinting, including three composite bioinks incorporating bioactive molecules . They crLikewise, Gao et al. co-printed nanoparticles of either bioactive glass (BG), hydroxyapatite (HA), or both BG and HA with bone marrow-derived human mesenchymal stem cells (hMSCs) suspended in a poly(ethylene glycol) dimethacrylate (PEGDMA) scaffold that was simultaneously polymerized to stimulate osteogenesis. In comparison to the other groups, hMSCs’ interaction with HA showed the highest cell survival and compressive modulus after 21 days in culture. The PEG-HA scaffold produced the most collagen, had the most alkaline phosphatase activity, and expressed the most collagen deposition, according to biochemical analyses. As a result, HA nanoparticles were more effective than BG in promoting hMSC osteogenesis in bioprinted tissues . Catros 2)/poly(ε-caprolactone) (PCL) porous scaffolds into ruthenium-loaded PEGylated liposomes (RL) to obtain a Ruthenium-loaded PEGylated liposome scaffold (RLS) composite as a result. The results show that the scaffold had a porous structure that is rather consistent and has good mechanical properties. Over the course of 48 h, the medication was released from RLS in a relatively consistent manner. RLS caused a mitochondrial malfunction, which lead to apoptosis in human osteosarcoma cancer cells (MG-63). All of the findings suggested that RLS could be a promising therapy option for osteosarcoma [2 for 10 min, nearly 90% of the cells were destroyed. While these materials have the potential for photothermal therapy, they can also be combined with a hydrogel matrix to stimulate the release of small molecule drugs when exposed to NIR wavelengths [Ye et al. developed a drug-release delivery system to inhibit the growth and recurrence of osteosarcoma by incorporating hydrophobically modified silica nanoparticles  were added to a polymeric ink containing oxidized alginate (OxA) and the mix was used to bioprint gels containing chondrocytes. The formation of reversible imine bonds between amines on the nanoparticles and aldehydes of OxA provoked considerably enhanced rheological properties resulting in the generation of porous constructs and an ear structure with overhangs and high structural fidelity. The improvement was mostly due to electrostatic interactions between cationic SiNPs and anionic polysaccharides and was significantly impacted by the size and concentration of the nanoparticles as well as the length of polymer chains . This stBakht et al. highlight recent achievements in the creation of functional nanocomposite hydrogels, focusing on their current and potential use as bioinks. This review discusses scientific progress in recent years, with a focus on bioinks with nanoparticles for regulated release of drugs and bioactive chemicals, as well as stimuli-responsive features with smart nanoparticles . Among tOverall, these studies indicate how such smart bioinks employing nanoparticles can promote differentiation and cell survival. Drug nanoparticles possess increased solubility and hence better bioavailability due to their very small size and wide surface area  as well 2+) and polyphosphate (polyP), reinforced with poly–caprolactone (PCL). The resulting granular PCL/Ca-polyP-MP hybrid material was used to 3D bioprint tissue-like scaffolds with open pores for cell migration using a layered architecture. The printed composite scaffold had biomechanical properties similar to cortical and trabecular bone. Staining for cell viability, cell density, and scanning electron microscopy (SEM) analyses revealed that this scaffold could attract and foster the growth of human bone-related osteosarcoma (SaOS-2) cells. Based on the findings, it was determined that granular PCL/Ca-polyP-MP hybrid material is ideal for the production of bioprintable scaffolds with morphogenetic potential as well as biomechanical stability [Microparticles, ranging in size from 1 to 1000 µm, can also serve as an effective tool for delivery, especially in the context of 3D bioprinting applications . These ptability . Finallytability .Microspheres are engineered materials defined as spherical or round-shaped microparticles . They ar2. Despite the second phase, CO2 was found to be able to reach the microspheres and sinter them, resulting in a mechanically robust structure that can endure substantial forces. Microsphere technology allows controlled release of encapsulated factors to enhance tissue growth through the design of unit cells within a scaffold, and the printing process with multiple printing heads allows focal placement of various phases to account for different needs of tissue to create scaffolds that serve the growth of both cartilage and subchondral bone, as required for tissue-engineered joint replacements [To make a highly viscous dispersion, Lohfeld et al. combined poly(lactic-co-glycolic acid) microspheres with agarose and hyaluronic acid hydrogel. To evaluate the effect of the fluid phase on sintering capabilities, the compounds were sintered with subcritical COacements . In paraacements . This stTo summarize, smart bioinks including microparticles and microspheres delivery systems are distinguished by certain characteristics such as (i) physical and chemical stability of the encapsulated active ingredient, which should be maintained throughout the process; (ii) simple, reproducible, and expandable manufacturing, ideally ending with optimal drug loading, maximum encapsulation, and maximum yield at the intended rate for an adequate time period; (iii) flowability and syringeabilitiy . 3O4 magnetic nanoparticles to their surface to render them magnetically responsive, then, human skin fibroblast cells were cultivated on arrays of the GelMA microstructures. Unlike prior rigid helical microrobots, the soft helical microswimmers were able to corkscrew over the step-out frequency while maintaining relatively high advancing velocity, indicating an unparalleled self-adaptive capability. GelMA microswimmers were also discovered to be highly cell compatible. They are also entirely degradable by collagenases, promote cell adhesion and development, and are gradually degraded throughout a culture by cell-released enzymes. These non-cytotoxic biodegradable hydrogel microswimmers are great prospects for several applications in medicine and tissue engineering as they reduce the worry of collecting microrobots after drug-release procedures [Magnetic helical microswimmers, also known as artificial bacterial flagella (ABFs), are microscale devices/robots that use rotating, oscillating magnetic fields, or magnetic field gradients to swim in liquid. They transform rotational motion into translational motion to perform 3D navigation in diverse liquids under low-strength rotating magnetic fields. ABFs microswimmers have been extensively researched as carriers for selective drugs and cells. Control of individual groups of swimmers within a swarm is required for numerous biological applications such as drug delivery and release or small-scale surgery in vivo and in vitro . Wang etocedures . Chemical composition and surface functionalization are frequently required for nanosized drug delivery devices, and techniques for their functionalization for targeted drug delivery are being widely used . While fHydrogels can regulate release performance by managing swelling or degradation thanks to their good compatibility and hydrophilicity . Photo-pHighley et al. made their microgels out of norbornene-modified hyaluronic acid (NorHA), poly(ethylene glycol) diacrylate (PEGDA), and agarose, and cross-linked them with a photoinitiator and light (NorHA and PEGDA) or cooling (agarose) . These t2+ cross-linked alginate, while the other layer is a stable network formed by F127DA photo-crosslinking. After Ca2+ was removed from the Na2CO3 solution, SMHs showed a high recovery ratio, and the elastic modulus remained practically steady after the shape memory cycle. In vitro investigations revealed that the drug release rate is faster than with typical drug-loaded hydrogels. The vitality of 3T3 fibroblasts was not affected, indicating its high biocompatibility. As a result, it is predicted that SMHs have a bright future as medication transporters and tissue engineering scaffolds [Wang et al. present a 3D bioprinting technology to fabricate the shape memory hydrogels with internal structure (SMHs) by combining sodium alginate and pluronic F127 diacrylate macromer (F127DA) using a dual network layout. One layer is a reversible network generated by Cacaffolds . Finallycaffolds . A colloid is a dispersion of one or more substances suspended in a liquid, forming a system with two separate phases . ColloidBhattacharyya et al. explained the parameter optimizations for semi-automated mixing of bioink components and 3D bioprinting with the twin-screw extruder head with alginate, alpha-tricalcium phosphate (α-TCP) micro/nanoparticles, and osteoblast cells . The TSEOverall, composite bioinks hold significant potential in engineering tissues due to the ability to create multifunctional bioinks . NumerouBioprinting is an additive manufacturing technology that uses bioinks in combination with cells to produce living structures . These bRecent advances in bioprinting provide a valuable tool to fabricate biomimetic constructs, which can be applied in different stages of drug release research. This paper presented many types of “smart” bioinks that can be used in 3D bioprinting. To encourage the desired behaviour of the cells being bioprinted, such bioinks use drug-releasing particles. After analyzing current research on this topic and identifying areas for further growth, it was concluded that “smart” bioinks and materials have the potential to revolutionize tissue engineering. For instance, in vitro experimental studies, though computational advances have resulted in more rationale computer-aided molecular design, still require gaining industry confidence and improving in vitro-in vivo correlations . Three-dFurthermore, considering the major advances in engineering and healthcare that 3D bioprinting has enabled, bioprinting in four dimensions (4D) has become an area of increasing focus. 4D printing occurs when a printed 3D item is exposed to external energy inputs such as temperature, light, or other environmental stimuli to trigger a change . 4D biop


The pandemic caused by SARS-CoV-2 (severe adult respiratory distress syndromeCoronavirus-2) and its most severe clinical syndrome, COVID-19, has dramaticallyimpacted service delivery in many radiology departments. Radiology (primarilychest radiography and CT) has played a pivotal role in managing the pandemic incountries with well-developed healthcare systems, enabling early diagnosis,triage of patients likely to require intensive care and detection of arterialand venous thrombosis complicating the disease. We review the lessons learnedduring the early response to the pandemic, placing these in the wider context ofthe responsibility radiology departments have to mitigate the impact ofhospital-acquired infection on clinical care and staff wellbeing. The potentiallong-term implications for design and delivery of radiology services areconsidered. The need to achieve effective social distancing and ensurecontinuity of service during the pandemic has brought about a step change in theimplementation of virtual clinical team working, off-site radiology reportingand postgraduate education in radiology. The potential consequences of thesedevelopments for the nature of radiological practice and the education ofcurrent and future radiologists are discussed. The virus isprimarily spread by contaminated droplets or aerosols from the respiratory tract ofinfected persons and by fomites – virus-contaminated residues of desiccateddroplets in the vicinity of an infected patient.2 Viable virus particles have also been recovered from thefaeces of infected patients,3 butevidence of faecal-oral transmission is scanty.The syndrome of pneumonia leading to respiratory failure now known as COVID-19 wasfirst described in Wuhan, China, in November 2019. Within weeks the causative agenthad been identified, assigned the name SARS-CoV-2,4 Maximal virusshedding by infected patients occurs in the prodromal or early symptomatic phases ofinfection, explaining the observed “super spreader” individuals andevents. It also exhibits longer prodromal and convalescent periods during whichpatients may transmit the infection. Its propensity to cause severe infection anddeath  is also anorder of magnitude greater than that of seasonal influenza.5SARS-CoV-2 is substantially more contagious than seasonal influenza (with which it isoften compared).6 This results incontamination of a wide area and a requirement for enhanced decontaminationprotocols. It also mandates the use of facemasks or breathing apparatus capable offiltering particles small enough to penetrate standard surgical facemasks (EN149FFP3 specification). Lists of AGPs published by public health agencies have focussedprimarily on those causing increased airflow across the upper respiratory tractepithelium,7 includinghigh-flow oxygen, mechanical ventilation and use of suction during dental surgeryand endoscopy of the airways and upper GI tract. Colonoscopy and CT colonographyhave not been classified as AGPs, although a study published in this issue of BJRoffers some evidence to the contrary.8 In a case control study comparing CT colonography with routinebody CT, the authors found evidence of airborne spread of coliform bacteria in morethan one-third of colonography examinations. The very limited evidence published todate suggests that the absolute risk of transmission posed by colonoscopy orcolonography is extremely low,9,10 but there is a need for further, larger scale research intothe epidemiology of SARS-CoV-2 transmission in the healthcare setting to mitigatefuture risk to health and avoidable disruption to healthcare delivery.11The risk posed by “aerosol-generating procedures” (AGPs) as a route oftransmission has been of particular concern. There is no universal definition of anaerosol, but the concept is that of virus-contaminated droplets small enough toremain airborne for an extended period, spreading further than 2 m from thesource.12Hospitals in countries with prior experience of Coronavirus epidemics (SARS andMERS) appeared better prepared to control nosocomial spread of infection, havingestablished protocols and better access to PPE, while large numbers ofinfections and deaths occurred among healthcare workers in countriesexperiencing a Coronavirus epidemic for the first time.11Initial containment efforts followed established guidelines and includedisolation or cohorting of infected patients, physical distancing of patients andstaff, use of personal protective equipment  and rigorous room and equipmentdecontamination.Nosocomial outbreaks among clinical teams threatened the sustainability ofservices as a result of staff absence due to illness or self-isolation. Therequirement to implement effective physical distancing, safe patient flows andeffective decontamination of equipment in radiology departments withovercrowded, cramped and inadequately ventilated accommodation necessitatedsubstantially reduced patient throughput. During the “first wave”of the pandemic, government-mandated curtailment of much out-patient activityand fear of nosocomial infection among patients resulted in a substantialreduction in cancer diagnosis and a rapid increase in those awaiting scheduledsurgical treatment.Much has been learned as a result of these early experiences. While thefundamental design and allocation of sufficient space to radiology departmentsis best achieved through strategic planning decisions, radiologists cancollaborate with infection control specialists to influence the reconfigurationof existing space and siting of new equipment so as to facilitate the adoptionof safely segregated workstreams, either on a permanent basis or as part ofresilience planning for future events. The case for investment in new imagingequipment, particularly CT scanners and point-of-care radiography and ultrasoundmachines, can only be strengthened by recent events.13,14has facilitated the safe restoration of diagnostic services.9,10 We must redouble effortsto optimise patient scheduling and flow by harnessing the capabilities ofradiology information systems and electronic patient records.15 This may include linkinghospital records with evidence of vaccination status. Close attention must bepaid to adequate provision of PPE and regular training in its correct use, as itis known to be highly effective in preventing cross-infection.16 Radiologists have a duty toadvocate for safe patient care as healthcare systems attempt to recover lostactivity; permitting excessive crowding of departments and unrealistic patientscheduling risks further outbreaks not just of COVID-19, but a range ofhospital-acquired infections.Efforts to contain SARS-CoV-2 have for the first time relied heavily on extensivetesting of asymptomatic patients and healthcare staff. The adoption of newpractices such as pre-appointment telephone screening of out-patients,reorganisation of departments and workflows and implementation of guidelinesdeveloped by the National Institute for Health and Care Excellence andspecialist societiesEarly in the pandemic, it became clear that social distancing requirements meantthat face-to-face clinical conferences and classroom-based radiology educationcould not continue. Video-conferencing, already established as a means ofenabling decision-making across multiple sites in a clinical network, has beensuperseded at unprecedented pace by cheaper, secure web-based applicationsutilising existing computer infrastructure, ushering in the “virtualmultidisciplinary meeting”. Within months, radiology training andcontinuing medical education providers have adapted or developed content toexploit these channels. Many hospitals have accelerated the roll-out ofteleradiology solutions for radiologists in response to staff demands to workremotely and the potential for an outbreak of infection to disrupt servicedelivery.These developments have met the immediate challenge of the pandemic, but theirconsequences for future radiological practice and education have yet to be felt.The ability to work from home, while attractive to those eager to achieve“life-work balance”, potentially diminishes the intangiblecontribution to patient care and job satisfaction arising from in-personinteraction with clinical colleagues and may result in reduced influence withinthe hospital. Existing multidisciplinary teams have functioned effectivelyonline during the emergency; it remains to be seen how well newcomers canintegrate into teams or how dysfunction in existing teams is resolved.The paradigm shift to online delivery of training and education has been welcomedby most trainees and practising radiologists. Challenges to the continuation oftraditional conferences, with their attendant travel and accommodation costs,will undoubtedly follow. On the other hand, online content will undoubtedlycontinue to improve, and offers opportunities to “level up” accessto the best educational content, as exemplified by Radiopaedia.org, which offersreduced-cost access to subscribers in low-income countries.13 Repeatedreports of outbreaks of infection due to failure of basic infection controlprotocol relating to ultrasound gel exemplify this,17 but numerous studies have demonstrated thepotential for infection to spread via contaminated machines, shared computerkeyboards, but above all as a result of inadequate hand hygiene.17,18While COVID-19 has naturally dominated recent thinking, it should not beforgotten that radiology departments have a shared responsibility to controlmany other microbiological hazards to patients and staff. The morbidity,mortality and costs of hospital-acquired infections have grown substantially inrecent years. This is a multifactorial problem: causes include the emergence ofmultidrug resistant bacteria, increasing numbers of patients with impaired hostdefences as a result of cytotoxic and immunomodulating drug therapies, andlapses in established infection prevention protocols. The modern radiologydepartment has become a “meeting point” where it is all too easyfor cross-infection to occur, and there is evidence that radiology departmentstaff may lack the knowledge and training to implement effective infectioncontrol practices.19 When faced with the global epidemic of humanimmunodeficiency virus (HIV) 35 years ago, healthcare staff rapidly embraced aset of “general measures” devised to prevent nosocomialtransmission of HIV  and other bloodborneviruses.20 TheCOVID-19 pandemic has reminded us of the potentially devastating consequences oflapses in infection control practice. We must respond by embracing a culture inwhich driving down the incidence of healthcare-acquired infections is seen asboth an organisational and a moral imperative. This will require increasedinvestment in staff training, acceptance of personal responsibility and rigorousenforcement of infection control policy.The measures required to achieve control of most microbiological hazards aregenerally simple and effective if applied correctly.


This expert opinion paper on cardiac imaging after acute ischemic stroke or transient ischemic attack (TIA) includes a statement of the “Heart and Brain” consortium of the German Cardiac Society and the German Stroke Society. The Stroke Unit-Commission of the German Stroke Society and the German Atrial Fibrillation NETwork (AFNET) endorsed this paper. Cardiac imaging is a key component of etiological work-up after stroke. Enhanced echocardiographic tools, constantly improving cardiac computer tomography (CT) as well as cardiac magnetic resonance imaging (MRI) offer comprehensive non- or less-invasive cardiac evaluation at the expense of increased costs and/or radiation exposure. Certain imaging findings usually lead to a change in medical secondary stroke prevention or may influence medical treatment. However, there is no proof from a randomized controlled trial (RCT) that the choice of the imaging method influences the prognosis of stroke patients. Summarizing present knowledge, the German Heart and Brain consortium proposes an interdisciplinary, staged standard diagnostic scheme for the detection of risk factors of cardio-embolic stroke. This expert opinion paper aims to give practical advice to physicians who are involved in stroke care. In line with the nature of an expert opinion paper, labeling of classes of recommendations is not provided, since many statements are based on expert opinion, reported case series, and clinical experience.The online version contains supplementary material available at 10.1007/s00392-021-01834-x. Whereas imaging of the brain and the brain-supplying arteries as well as electrocardiogram (ECG) monitoring is standard in stroke diagnostics , 2, discAbout one out of five strokes is cardio-embolic in nature and stroke-recurrence rates are comparably high after cardio-embolic stroke. A cardiac source of embolism is more likely if multiple acute or subacute strokes in different vascular territories are detected, secondary hemorrhagic transformation is observed or a Valsalva maneuver preceded symptom onset . There aThis expert opinion paper collates and weighs the prevalence of stroke-related cardiac pathologies and most commonly used cardiac imaging methods with a focus on left atrial imaging. Summarizing present knowledge, expert-based suggestions for practical application of cardiac imaging after ischemic stroke or TIA are provided. Using the Delphi method, experts answered questionnaires in two rounds. Given (key point) recommendations are labeled as follows: ** if all experts agreed; * if the vast majority (≥ 90%) of experts agreed; without * if the majority (≥ 60–89%) of experts agreed.Cardiac and aortic sources of cerebral embolism resulting in stroke/TIA are heterogeneous . A potenTreatment strategy changes by the initiation of anticoagulation have been reported in 3.4–8.0% , 38.The most widely used imaging technology in post-stroke patients is echocardiography, which can be performed by TTE or TOE. TOE usually has a higher accuracy for the detection of potential cardio-embolic sources. Due to the anatomical neighborhood of heart and esophagus the TOE probe comes close to the heart permitting use of high-frequency ultrasound probes (4.2–7.4 MHz) enabling a high spatial and temporal image resolution , 39. TheTOE is a safe examination if rare contraindications are respected . In manyTTE is non-invasive and well suited for the assessment of the morphology and function of cardiac cavities, especially the left ventricle and left atrium, as well as for the assessment of hemodynamically significant valvular pathologies. Contrast-enhanced TTE with ultrasound agent opacification of the cavities of the left heart can improve endocardial delineation, exclude left ventricular thrombi and support ischemia diagnosis during stress echocardiography. It should be performed in patients with poor acoustic windows or the suspicion of a left ventricular thrombus. TTE is limited by impaired ultrasound penetrance, especially in obese individuals or patients with pulmonary disease.Key pointsEchocardiography is feasible and safe in patients with (acute) ischemic stroke or TIA.**TTE and TOE have to be considered as complimentary methods that provide distinct aspects of cardiac pathologies.**TOE is the method of choice for refined imaging of the atria and the interatrial septum. Details of (peri-)valvular abnormalities can be diagnosed with higher accuracy.**TTE is able to assess global and regional wall motion, ventricular abnormalities and left atrial function.**Systematic imaging and documentation of a comprehensive, standardized echocardiography work-up is illustrated in Fig. With the rapid advancement of imaging methods with increasingly high resolution and short image acquisition duration, other modalities such as cardiac CT/CT angiography as well as cardiac MRI have become available for diagnostics after stroke. A semi-quantitative comparison of the different imaging modalities and specific indications is provided in Table ECG-gated cardiac scanning with multi-detector 64-slice CT systems that scan large volumes at high speed (short breath hold) the temporal and spatial resolution significantly improves and enables visualization of small and volatile structures . The useFurthermore, cardiac CT can contribute to the differentiation of left ventricular thrombi and other intraventricular masses due to its higher spatial resolution and improved delineation of endocardial borders, but CT usually follows initial evidence from an echocardiogram in the clinical routine , 52. TheKey pointsCardiac CT is feasible in stroke patients due to the short acquisition time; however, it comes at the cost of radiation exposure.*Cardiac CT can be a complementary imaging method for additional work-up of specific patients with acute ischemic stroke or TIA, but is not optimal for the evaluation of most frequent cardiac sources of embolism.*Cardiac MRI is emerging as a non-invasive tomographic imaging method for post-stroke cardiac work-up , 65. TodAfter initial TTE, cardiac MRI can further aid to differentiate unclear, less common cardiomyopathies, e.g. in case of cardiac amyloidosis or non-compaction cardiomyopathy –70. In aThe feasibility of cardiac MRI in consecutive stroke patients has been demonstrated in a single-center study as 89 of 103 patients with acute ischemic stroke completed the 50-min examination . The useKey pointsCardiac MRI is a complementary imaging method for work-up of specific cardiac pathologies, for example tumors, cardiomyopathies and left ventricular thrombi or prior myocardial infarction.**Cardiac MRI is not optimal for the evaluation of PFO or the detection of endocarditis.In general, cardiac MRI requires comparatively long image acquisition times and affords the patient to follow commands, limiting the feasibility in acute stroke patients.*The importance of left atrial imaging increases as the concept and clinical relevance of atrial (cardio-) myopathy evolves. Atrial cardiomyopathy is defined by the presence of structural, architectural, contractile or electrophysiological changes and may carry an increased risk of embolic stroke independent of (ECG detected) AF , 74. AnaKey pointsLeft atrial imaging may be used to assess atrial cardiomyopathy.*Presence of atrial cardiomyopathy can help to tailor the intensity of post-stroke monitoring for AF.**Careful work-up is necessary to identify potential cardiac sources of embolism. Furthermore, stroke work-up provides an opportunity to screen for cardiac comorbidities that may prompt further diagnostic evaluation and affect treatment that improves cardiovascular outcome.In Germany, there is broad consensus to perform echocardiography in selected patients with acute ischemic stroke, as registry data demonstrated an increased use of echocardiography from 62.2% in 2001 to 74.0% in 2006 . This isAn optimal secondary stroke prevention strategy may also affect health economics. However, no prospective randomized study has proven that secondary prevention measurements  are efficacious with regard to clinically relevant endpoints. Furthermore, there is only limited data available on how often echocardiography leads to relevant changes in therapeutic management. Observational studies, mainly focusing on changes in secondary stroke prevention, suggest that this is only rarely the case, rendering a reduction of stroke recurrence or reduction of other (vascular) endpoints unlikely , 96, 97.An interdisciplinary team routinely collaborating on the cardiology work-up of post-stroke patients may facilitate cross-discipline communication and improve patient care. Ideally, a specialized cardiovascular imaging suite is nearby or even integrated in the stroke unit, which permits short transportation times or bedside echocardiography. If post stroke echocardiography is performed in a cardiology routine environment, fixed slots for stroke unit patients may facilitate logistics. Echocardiographic work-up should ideally be finalized during the hospital stay to ensure an interdisciplinary approach for patient discussion and potential further diagnostic or therapeutic decisions. Therefore, adequate structures and resources are necessary for stroke-care hospitals. In case of limited echocardiographic capacities and short duration of in-hospital stays, echocardiographic work-up can be performed post-discharge in patients in stable cardiac conditions with an established non-cardiac cause of stroke.The quality of echocardiographic findings is investigator-dependent , 102. ThKey pointsA standardized set-up and experienced investigators are needed for echocardiographic examinations in stroke patients, which should follow a systematic, standardized protocol.*standard operating procedures for post-stroke cardiac imaging may enhance decision making and advise further work-up including special cases where cardiac CT or MRI are required.*An interdisciplinary team and Cardiac imaging should be performed if it has potential therapeutic consequences. In case of a defined non-cardiac source of stroke  and a low cardiovascular risk profile, cardiac imaging is not essential after stroke or TIA.Since guidelines recommendation consistently encourage echocardiography in patients with suspected embolic stroke and without contraindications for oral anticoagulation, this approach selects in particular younger patients with stroke or TIA. As cardiac comorbidities such as coronary heart disease, left ventricular hypertrophy, and valvular dysfunction naturally becomes more common in older age groups, expert consensus is that TTE should be considered in patients with at least one established cardiovascular risk factor Fig. .Fig. 4ExAcute ischemic stroke or TIA is an indicator of a comparably high risk of cardiovascular co-morbidities that may not be directly causally related to the present stroke but may significantly affect stroke recurrence rate and survival . In partKey pointsStroke or TIA patients should undergo cardiac imaging, if stroke etiology is uncertain.*Stroke or TIA patients should undergo cardiac imaging, if the presence of pathological findings would alter  management.*Cardiac imaging should be considered in ischemic stroke patients with at least one established cardiovascular risk factor to identify cardiac comorbidities, unless cardiologic work-up within the last 6 months is documented in stroke patients without signs of cardiac dysfunction.Stroke or TIA patients with AF should undergo cardiac imaging for further cardiac work-up, if the presence of a left-sided atrial or ventricular thrombus would alter  management or if a first episode of AF was documented in-hospital.*Whereas routine echocardiography in all stroke patients is not recommended, guidelines consistently encourage echocardiography in patients with suspected embolic stroke with unsuspicious neurovascular imaging and no contraindications against anticoagulation where a diagnosis would lead to a treatment change , 6, 8, 9For the diagnosis of atrial sources of emboli TOE is necessary. However, in the vast majority of cases, patients with cardiac thrombi have underlying functional or structural cardiac alterations . If any of these conditions is absent, the 12-lead ECG is normal, and no history of cardiovascular disease is present left atrial thrombi are a rare finding , 31, 116Increased left atrial dimensions or parameters of reduced left atrial function summarized as atrial cardiomyopathy may hint towards paroxysmal AF as a potential cause of stroke, reduced left ventricular ejection fraction, and regional wall motion abnormalities towards left ventricular thrombus , 73, 119In stroke patients with known AF prior to stroke, a TTE should be considered . Furthermore, a TOE should be performed in stroke patients with AF, if the presence of an atrial thrombus would alter medical management, e.g. lead to immediate oral anticoagulation despite the risk of secondary hemorrhagic transformation.Cardiac CT imaging is suitable for the diagnostic classification of patients with suspected coronary artery disease and cardiovascular risk stratification –60. HoweFor now, it appears that cardiac CT and MRI may serve as complementary imaging modalities in clinical studies or for in-depth work-up in case of suspected cardiac abnormalities that remain inconclusive in echocardiographic examinations. CT/MRI can also be applied in rare cases where contraindications against TOE are present or image interpretability is impaired. The use of either method will not only depend on the local expertise but largely on the availability of high-quality imaging facilities. One may argue that chest x-ray is routinely accessible in the stroke unit setting, and can reveal cardiac enlargement, pulmonary congestion, pleural effusion and even valve calcifications. As no respective studies are available , it is eKey pointsEchocardiography is the gold standard imaging modality after ischemic stroke or TIA, if a pathological finding would alter  management.*TOE and TTE should be performed (preferably in a single session) if a cardio-embolic source is deemed a probable cause of ischemic stroke or TIA.TTE should be performed in patients with suspicious findings during TOE, if TOE alone is not sufficient for the comprehensive evaluation of the specific pathology .TOE should be performed in patients with suspicious findings during TTE, if TTE alone is not sufficient for the comprehensive evaluation of the specific pathology (e.g. endocarditis or assessment of (inter)atrial structures).**TOE should be performed (in patients with so far cryptogenic stroke aged 16–60 years), if PFO presence would alter further management.*TTE should be performed in stroke patients with a first episode of AF in-hospital.*TTE should be considered in ischemic stroke patients with at least one established cardiovascular risk factor unless recent cardiologic work-up is documented.*Cardiac CT should be considered, if there is a suspicion of cardiac or extra cardiac abnormalities, based on other imaging modalities, which are assumed to be clinically relevant. As cardiac CT is associated with radiation exposure, use of cardiac CT should be based on an individual decision.*Cardiac MRI should be considered, if there is a suspicion of left ventricular thrombus or cardiac tumor or in case of an unclear cardiomyopathy after contrast enhanced echocardiography.With intensified cardiac imaging, work-up of incidental findings needs to be considered. Such findings may result in uncertainty on diagnostic and treatment consequences in often older and multi-morbid patients. In stroke patients a PFO is a frequent  finding due to the high prevalence in the general population . FrequenKey pointsDiagnostic algorithms and interdisciplinary standard operating procedures should be established to avoid over-diagnosis and “over-treatment” because of incidental findings of cardiac imaging.*Overall, cost-effectiveness analyses are impaired by the limited data on the effectiveness of treatment for most imaging findings. At present, reliable numbers exist for left atrial appendage thrombus and subsequent OAC only and will probably soon be available for PFO closure . WhereasFor other imaging methods such as cardiac CT or MRI implementation in acute stroke imaging protocols has been suggested, but robust data on the benefit beyond echocardiography are not available. Cardiac CT and in particular MRI examinations are significantly more expensive than echocardiography, with TTE being the cheapest of all cardiac imaging methods . For allKey pointsCost-effectiveness of systematic cardiac imaging in stroke patients should be addressed in prospective trials.Aortic arch atheromas have been related to recurrent embolic stroke . A CT scKey pointsA CT angiography scan can visualize the whole aortic arch and the descending aorta whereas TOE often is impaired by suboptimal acoustic properties of the aortic arch.MRI angiography can depict aortic pathology but acquisition is time-consuming, more expensive, and limited with regard to calcifications, mobile structures and ulcerations.*In conclusion, stringent high-quality cardiac imaging in ischemic stroke or TIA patients offers the opportunity to reveal stroke causes with direct therapeutic consequences and to improve global cardiovascular risk assessment in a high-risk population for cardiovascular disease. According to the working group, TTE should be considered in stroke patients with at least one established cardiovascular risk factor. A TOE should be performed, if a cardiac source of embolism is suspected or TTE findings need further work-up. Non-invasive cardiac imaging using CT or MRI can be viewed as complementary methods, but should currently be restricted to specific diagnostic questions and clinical studies.Supplementary file1 (DOCX 75 kb)Below is the link to the electronic supplementary material.


The coronavirus disease 2019 (COVID-19) public health crisis has reached critical mass, but interdisciplinary research efforts have provided the global community with the first effective medical intervention to fight the pandemic—COVID-19 vaccines. Two of the vaccines approved for use in the United States and Europe deliver nucleic acid in the form of mRNA, the success of which would not be possible without biomaterials. Lipid nanoparticle (LNP)-based mRNA vaccines, discussed in this perspective, protect nucleic acids from degradation and deliver cargo directly to the intracellular compartment of cells where it is translated into the antigenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein that triggers protective immune responses. Despite success of LNP-based mRNA vaccines thus far, the pandemic has highlighted the need for emerging technologies that enable rapid development and increased accessibility to vaccination. Microneedle arrays, also discussed in this study, provide features that could lower barriers to vaccine access in resource-poor regions. The ability to exchange antigens within arrays could also facilitate swift vaccine deployment as public health needs evolve . Therefore, the COVID-19 pandemic has spotlighted the readiness and value of biomaterials for the prevention and management of disease outbreaks. In just <2 years, there have been 250+ million confirmed cases and >5 million deaths worldwide . Studies examining postacute sequelae of the disease reveal that 20% of hospitalized COVID-19 patients are discharged with a new disability  vaccines. In 2020, Pfizer-BioNTech and Moderna's COVID-19 vaccines  . Instead of delivering viral antigen, they deliver part of the viral genetic code in the form of mRNA or DNA that contains instructions for synthesis of viral protein. Once the target antigen is identified by sequencing the viral genome of interest, nucleic acids are synthetically produced for inclusion in vaccine formulations  -based FDA-approved product for the treatment of transthyretin amyloidosis  positively charged ionizable lipids, (2) polyethylene glycol that increases particle persistence by reducing binding of proteins designating them for clearance, (3) stabilizing agents, such as cholesterol, and (4) naturally occurring phospholipids  for long-term storage. This posed logistical hurdles for distribution. In contrast, Moderna's vaccine can be stored at −20°C. The current view is that the susceptibility of naked mRNA to hydrolysis dictates strict cold storage conditions. Although proprietary, one can speculate that specific LNP formulations allow for relaxed storage requirements. For example, if the mRNA is located inside the nanoparticle core together with cationic lipids that enhance its stability , Zika virus (NCT04064905), and Rabies virus (NCT03713086), among others , microneedle arrays (MNAs) are one existing biomaterials-based vaccination strategy whose development has been accelerated by the current pandemic , loading capacity, and structural stability. Dissolvable microneedles are the current front-runner in design. They are manufactured using biodegradable polymers encapsulating large vaccine doses compared with solid microneedles. Because they are degradable, they offer the additional benefit of preventing inappropriate or accidental reuse  that traffic to lymph nodes to initiate immune responses , irradiation, and several freeze–thaw cycles . Importantly, they benchmarked their MNA vaccine against a subcutaneous bolus injection and demonstrated that mice exhibit comparable T cell responses , the U.S. government has provided $1.9 million to three groups  for the development of microneedle skin patches for preventing COVID-19 . No human data from clinical trials are available yet, so it remains to be seen whether scaffold-based COVID-19 vaccinations will translate. Nonetheless, these investments demonstrate the promise of MNAs as a tool for developing globally accessible vaccines (Arya and Prausnitz, Even if MNAs do not impact the immediate crisis, research into the utility of this vaccine platform spurred by the COVID-19 pandemic will further development of this biomaterials platform that could be adapted to address emerging public health needs.Scientists and engineers took advantage of the features of LNPs to rationally engineer efficacious mRNA vaccines that altered the course of the pandemic. This also represents a monumental achievement for the biomaterials field and the future of biomaterials-based clinical translation. Early preclinical data from the emerging MNA vaccine delivery platform suggests that desirable features such as self-administration and the ability to rapidly exchange antigens relevant for public health crises may address deployment hurdles in resource-poor areas. Together, these creative technological advances in biomaterials-based vaccine research represent a silver lining of the pandemic response.


There was also an indirect relationship between one form of impulsivity and crashes through risky driving . Additional analyses did not find an independent contribution to crashes for frequent cell phone use. These results suggest a pattern of risky driving practices associated with impulsivity in young drivers, indicating the benefit of exploring a more comprehensive safe driving strategy that includes the avoidance of cell phone use as well as other risky practices, particularly for young drivers with greater impulsive tendencies.This study examines whether cell phone use stands apart from a general pattern of risky driving practices associated with crashes and impulsivity-related personality traits in young drivers. A retrospective online survey study recruited 384 young drivers from across the United States using Amazon’s Mechanical Turk to complete a survey measuring risky driving practices (including cell phone use), history of crashes, and impulsivity-related personality traits. Almost half (44.5%) of the drivers reported being involved in at least one crash, and the majority engaged in cell phone use while driving (up to 73%). Factor analysis and structural equation modeling found that cell phone use loaded highly on a latent factor with other risky driving practices that were associated with prior crashes ( In North America, both epidemiological and observational studies have shown that cell phone use while driving is associated with increased crash and near-crash risk ,2. In 20One possibility is that drivers who frequently engage in cell phone use while driving may also engage in other intentional risky practices while driving . A 2013 There is also evidence of a link between impulsivity-related personality traits, specifically, weaker impulse control (acting without considering consequences) and sensation seeking (seeking novel and exciting experiences) and risky driving practices, including cell phone use, in young drivers ,14. In aThere are well-documented developmental changes in impulsivity-related personality traits that may explain increased risk-taking behaviors in young drivers ,21,22,23In order to better understand risky driving practices, the current study recruited a sample of young adult drivers from across the United States to determine: (i) whether cell phone use while driving is only one of several other co-occurring risky driving practices in which young drivers engage (replicating prior findings); (ii) if this pattern of dangerous driving behavior is related to crashes (maintaining constant factors such as sex and number of years driving); and (iii) what role sensation seeking and impulsivity play in the relationship between risky driving and crashes. Thus, this study expands on our prior work by including a larger and more geographically diverse sample, with the addition of other known risk factors of impulsivity-related personality traits. We hypothesize that (i) drivers who frequently engage in cell-phone use while driving will also frequently engage in other risky driving behaviors associated with crashes, and (ii) that this pattern of risk-taking will be associated with weaker impulse control (due to the ongoing maturation of executive cognitive control at the transition to adulthood). If a pattern of risky driving is associated with a history of crashes and risk-taking personality differences, then this has important implications for future research to inform the design and delivery of interventions at both the individual and public health level, potentially calling for a more personalized prevention strategy.Participants were recruited via the Amazon MTurk Prime Panels and directed to an online survey hosted by the Psytoolkit survey platform. A sample of 384 licensed drivers aged 18–24 years from across the United States completed the survey, and all data were collected over the course of 5 days in November 2017: the mean age was 21.41 years (SD = 1.96). The survey consisted of questions capturing self-reported crash and citation history and risk-related driving practices. In addition, 3 quality assurance questions were included that asked participants to respond in a specific way in order to measure attention to the survey . If two questions were answered incorrectly, that participant’s data would be removed from analyses. No participants were excluded from the analysis for lack of attention. History of crashes was recorded as in our prior work  . AdditioThe survey also included self-report measures of sensation seeking and acting-without-thinking, a form of impulsivity. Sensation seeking was measured with 4 items from the Brief Sensation Seeking Scale , with reCorrelations between each risky driving practice and crashes were examined, along with other variables. A confirmatory factor analysis was used to test a two-factor versus a single-factor structure of risky driving practices identified in a prior sample . First, n = 281, 73.2%) reported engaging in cell phone calls while driving, and 237 (61.7%) reported answering a text message while driving. Several of the behaviors were related to experiencing a prior crash apart from cell-phone use . In other aspects of risk-taking, 15 drivers (3.9%) reported never or rarely wearing a seat belt as a passenger, and 7 (1.8%) reported never or rarely wearing a seat belt as a driver, which is comparatively lower reported risk-taking with regard to seatbelt use compared to national observations on these same survey items ) and sensation seeking  remained while controlling for the other variables. In addition, risky driving was related to acting-without-thinking . There was an indirect relationship between acting-without-thinking and crashes through risky driving , indicating that persons engaging in risky driving were also more likely to act without thinking.The model was modified to test for any indirect effect of acting-without-thinking on crashes through risky driving (using 1000 bootstrap samples), while removing the direct relationship between acting-without-thinking and crashes (Crash Model 2). This model fit the data well . b = 0.45,  and the indicator representing high risk on both risky driving and cell phone use  was not associated with prior crashes. These findings support the hypothesis that cell phone use while driving is but one of several risky driving practices in young people that may contribute to crashes.These results replicate those of , and corTaking these two findings together, future research could guide interventions that address the driver rather than the behavior. For example, when drivers are identified as at-risk based on individual characteristics  or behaviors , the avoidance of cell phone use as well as other risky practices could be included in driving countermeasures. Clinic-based brief interventions could expand the model developed for targeting young drivers who present with injuries after alcohol-related crashes ,34. ThesThe driving practices and crash outcomes in the current (and prior) study relied on self-report responses. However, the risk of social desirability bias may have been reduced by the anonymous recruitment of participants and online survey. Furthermore, prior work has also relied on self-reported crashes (which can be over- or under-reported) ,36,37, iThis study relied on Mechanical Turk as a recruitment method, which might be subject to recruitment bias; however, there is evidence to suggest that MTurk samples are more diverse than other online survey samples and American campus samples . FurtherA further limitation of this work is that the characterization of cell phone engagement was limited and did not specify if the call was hand-held or not, nor did we ask about the use of any other cell phone features while driving , or other forms of distracted driving . In addition, trends in phone use while driving among young drivers may have changed since these data were collected in 2017. Future work should consider including more items addressing contemporary risky driving practices to capture cell phone distractions while driving.These findings support prior research to suggest that cell phone use while driving is part of a pattern of risky driving associated with crashes that is also associated in part with differences in acting-without-thinking impulsivity. Furthermore, current point-of-care brief interventions  could consider broadening their scope from addressing one risky behavior. When these drivers are identified as engaging in any one of these dangerous practices, there is an opportunity to deliver interventions that also address related risk behaviors when being treated for injuries after a crash or when pulled over following a citation for dangerous behavior. Assessments of personality traits related to crashes may also help in the early identification of drivers more at risk, who may stand to benefit most from targeted interventions.


To evaluate the impact of the coronavirus disease 2019 (COVID-19) pandemic and the subsequent implementation of tuberculosis response measures on tuberculosis notifications in Zambia.We used an interrupted time-series design to compare monthly tuberculosis notifications in Zambia before the pandemic (January 2019 to February 2020), after implementation of national pandemic mitigation measures (April 2020 to June 2020) and after response measures to improve tuberculosis detection (August 2020 to September 2021). The tuberculosis response included enhanced data surveillance, facility-based active case-finding and activities to generate demand for services. We used nationally aggregated, facility-level tuberculosis notification data for the analysis.Pre-pandemic tuberculosis case notifications rose steadily from 2890 in January 2019 to 3337 in February 2020. After the start of the pandemic and mitigation measures, there was a −22%  immediate decline in notifications in April 2020. Larger immediate declines in notifications were seen among human immunodeficiency virus (HIV)-positive compared with HIV-negative individuals . Following roll-out of tuberculosis response measures in July 2020, notifications immediately increased by 45% (95% CI: 38 to 51) nationally and across all subgroups and provinces. The trend in notifications remained stable through September 2021, with similar numbers to the predicted number had the pandemic not occurred.Implementation of a coordinated public health response including active tuberculosis case-finding was associated with reversal of the adverse impact of the pandemic and mitigation measures. The gains were sustained throughout subsequent waves of the pandemic. All individuals diagnosed with drug-susceptible tuberculosis and registered for treatment initiation (notified) were included in the analysis, regardless of age or tuberculosis type .For the analysis we used nationally aggregated, routinely captured tuberculosis programme data supplied by the National Tuberculosis and Leprosy Programme. Every health facility in Zambia uses a paper-based system to document tuberculosis case notifications. Once an individual with tuberculosis starts treatment, their data are recorded in a facility-level register: demographic characteristics , tuberculosis laboratory results (such as the Xpert® MTB/RIF assay or smear microscopy status), and diagnosis and treatment start dates. Every month, facilities send tuberculosis notification data to the district, where they are aggregated. Data are then sent to the province, where they are aggregated again before being sent to the National Tuberculosis and Leprosy Programme. Next, a monitoring and evaluation team reviews the data; any incomplete or irregular data are clarified and corrected before undergoing final national-level data aggregation. To illustrate the relationship with COVID-19 infections over the same period, we also used publicly available data on the daily number of COVID-19 cases reported in Zambia.This study was a population-level analysis without the use of any patient identifiers. The University of Zambia biomedical research ethics committee reviewed the study and classified it as exempt from human subjects’ research review.Measures to mitigate the risk of COVID-19 transmission were implemented nationally in late March 2020. Mitigation measures comprised travel restrictions, closures of restaurants, bars and educational institutions, and limitations of public gatherings to less than 50 people. While many of these measures had been scaled down by September 2020, most individuals in Zambia resumed their normal daily activities by August 2020, in large part because they could not afford the direct and indirect costs of continued compliance with the measures. As the pandemic progressed, mitigation measures were scaled up and eased in response to subsequent surges in COVID-19 infections .11During the initial period in which pandemic mitigation measures were in place in Zambia, tuberculosis diagnostic and treatment services remained open throughout the country. Adaptations to existing tuberculosis services were made to protect vulnerable patients and staff and avoid overcrowding at facilities, including pausing community-based activities, providing prolonged tuberculosis medication refills, and undertaking telephone-based household contact tracing and tuberculosis treatment assessments. HIV services made similar adaptations. HIV services already provided extended multi-month dispensing of antiretroviral therapy (ART) for up to 6 months to clinically stable patients. This strategy was therefore substantially scaled up across Zambia at the onset of the pandemic.In July 2020, following 3 months of declining tuberculosis case notifications (April to June), the National Tuberculosis and Leprosy Programme worked with several stakeholders to design and begin implementing a multicomponent strategy to improve tuberculosis surveillance and tuberculosis detection during the ongoing COVID-19 pandemic .Box 1Each week, staff at the National Tuberculosis and Leprosy Programme held an online meeting to review national tuberculosis notification data. Participants were staff from implementing partner organizations and those involved in tuberculosis programming from the facility level through to the national level. Centralized teams and higher-performing facilities and districts (selected based on comparisons to set tuberculosis notification targets) were able to share best practices and provide support to lower-performing facilities and districts. To support this initiative, the National Tuberculosis and Leprosy Programme developed a weekly data reporting tool; built local capacity among facility-based health-care workers to improve record-keeping at all patient entry points; and began reporting data weekly rather than monthly. The permanent secretary of the health ministry officially launched this initiative to enhance support by leadership at each level. Since the initiative’s launch in July 2020, weekly meetings have been sustained throughout the pandemic.Beginning in July 2020, we designed a strategy to motivate public health facilities across all provinces to begin systematic symptoms-based screening of all individuals attending the facility for active tuberculosis disease, regardless of the reason for presentation. We initially prioritized facilities contributing 80% of notifications at the district level. Activities included (i) training and reorienting health-care staff at each facility using standard operating procedures and standardized training slides; (ii) increasing the availability of digital chest X-rays for use as a triage test; and (iii) improving access to urine tuberculosis tests . Over the subsequent months, we scaled up the number of tuberculosis diagnostic facilities providing facility-based active case-finding. Once saturation was achieved at diagnostic facilities, we introduced systematic, symptoms-based screening at tuberculosis treatment-only facilities (those with no tuberculosis diagnostic capacity). Sputum samples were collected from patients who screened positive for tuberculosis and sent to local diagnostic facilities for microbiological testing. We gave technical supervision, support and mentorship around this initiative to individual health facilities through weekly provincial-level tuberculosis situation room meetings. Since initial scale-up, all facilities that we have trained have sustained systematic symptoms-based screening throughout the pandemic.Initially, community-based volunteers were working within health facilities to generate demand for tuberculosis services among community members already attending health facilities for any reason. After initial COVID-19 mitigation measures were scaled down in September 2020, we extended these activities to communities to raise further awareness about tuberculosis and COVID-19 and to encourage people to seek care when symptomatic. We developed a standardized guide to support these sensitization activities, focused on (i) raising awareness about tuberculosis symptoms and risks; (ii) educating individuals to request tuberculosis screening; and (iii) providing information about where tuberculosis screening and testing was offered.To safely resume home visits, we provided community-based health-care workers with refresher training during lunchtime meetings, using standardized training guides. Trainings were led by staff of the health ministry and implementing partner organizations. Community-based health-care workers were provided with specific education on COVID-19 infection and prevention control measures and were also supplied with washable masks and hand sanitizer.COVID-19: coronavirus disease 2019.We undertook an interrupted time-series analysis using an ordinary least-squares segmented linear regression model. We used the Cumby–Huizinga general test and autocorrelation plots (up to lag order = 12) to assess for autocorrelation and possible seasonality. We used Newey–West standard errors to account for autocorrelation. The user-written ITSA command within Stata version 17.0  was used to conduct interrupted time-series analyses.We defined three time periods for all interrupted time-series analyses: before the pandemic ; during the pandemic, at the start of pandemic mitigation measures ; during the pandemic, after implementation of tuberculosis case-finding measures . March 2020 and July 2020 represented phase-in periods and were not included in the data analysis. For March, the first COVID-19 cases were announced on 18 March 2020 and transmission mitigation measures were put into place later that month. For July, the scale-up of active tuberculosis case-finding activities across provinces did not begin until later in the month. Therefore, the effects of such measures on tuberculosis notifications in March and July, respectively, may not have been apparent until the following complete calendar month. Thus, two interruptions occurred between February and April 2020 (interruption 1) and between June and August 2020 (interruption 2).We estimated several pre- and post-interruption measures of monthly tuberculosis case notifications and corresponding 95% confidence intervals (CI), including: (i) baseline trends in tuberculosis notifications before the pandemic (Period 1 trend); (ii) the absolute number of tuberculosis notifications and the relative per cent difference  in notifications immediately following interruptions 1 and 2 (change in level); (iii) the absolute trend in tuberculosis notifications during Periods 2 and 3 and the trend in these periods relative to the previous period (change in trend for Periods 2 and 3).We also estimated the predicted number of tuberculosis case notifications in September 2021 had the pandemic not occurred and had the subsequent tuberculosis response measures not been implemented  and compared this with the number of tuberculosis case notifications predicted for September 2021 after accounting for both interruptions . We conducted all analyses overall  and according to age , HIV status , tuberculosis classification type  and by province. We wanted to evaluate whether possible impacts on tuberculosis notifications differed by sex, but due to the way routine data are aggregated and reported in Zambia, monthly sex-disaggregated data were not available for interrupted time-series analyses. We therefore descriptively assessed quarterly sex-disaggregated notification data to evaluate whether differential impacts by sex may be present.Before the pandemic started in Zambia, tuberculosis notifications were slowly increasing by about 32 cases (95% CI: 22 to 42) notified per month, from a baseline of approximately 2890 cases (95% CI: 2804 to 2975) in January 2019 to 3337 cases (95% CI: 3252 to 3421) in February 2020 ; Fig. 1.The relationship between daily COVID-19 cases and monthly tuberculosis notifications is shown in In the three months following the start of the pandemic and after a large initial decline in national tuberculosis notifications, notifications remained steady on a month-to-month basis and did not substantially differ from pre-pandemic monthly trends ; Fig. 1.In July 2020, the National Tuberculosis and Leprosy Programme began rolling out several tuberculosis response measures in all provinces to bolster tuberculosis detection. The following month (August 2020), national tuberculosis notifications immediately increased from a predicted 2700 cases (95% CI: 2603 to 2796) to an observed 3906 cases (95% CI: 3761 to 4051), a rise of 1206 cases (95% CI: 1038 to 1375) and a 45% (95% CI: 38 to 51) increase . ImmediaThe trend in monthly national tuberculosis notifications between August 2020 and September 2021 remained steady . This figure was not significantly different to the estimated number of notifications assuming a continuation of pre-pandemic trends  in March and early April 2020.We implemented enhanced tuberculosis response activities, scaled up facility-based active case-finding measures, and increased access to improved tuberculosis diagnostic tools. These activities appeared to be associated with a marked improvement in tuberculosis notifications during the pandemic, and as the activities were sustained, so too was their positive impact on notifications as the pandemic continued. Due to our quasi-experimental study design, we cannot discern whether improvements in tuberculosis notifications are the sole result of these activities or may be due to other secular trends.The strengths of this analysis include the use of routine, national-level programmatic data and of disaggregated analyses among several key subgroups and in all Zambian provinces. This method allowed us to assess for possible differential trends in tuberculosis notifications before and during the COVID-19 pandemic. Furthermore, we analysed notification trends across three periods covering 33 months, including multiple waves of the pandemic, which allowed us to determine that gains in notifications following implementation of tuberculosis response activities were sustained. There were some limitations, however. We were unable to determine what factors underpinned the immediate decrease in tuberculosis notifications in Zambia following the emergence of COVID-19 cases. However, the change likely reflects both individual- and health-system-related factors. For example, individuals may have been reluctant to seek care for their symptoms due to fear or stigma.In conclusion, the COVID-19 pandemic and the associated mitigation measures had a substantial impact on tuberculosis case notifications in Zambia. A carefully coordinated public health response, including active tuberculosis case-finding strategies, was feasible to implement and was associated with a return of tuberculosis case notifications to pre-pandemic levels. The gains were sustained throughout subsequent waves of the pandemic. Continued vigilance will be required during the ongoing pandemic to ensure high tuberculosis diagnosis and treatment coverage levels.


The majority of cellular DNAs in eukaryotes are organized into linear chromosomes. In addition to chromosome DNAs, genes also reside on extrachromosomal elements. The extrachromosomal DNAs are commonly found to be circular, and they are referred to as extrachromosomal circular DNAs (eccDNAs). Recent technological advances have enriched our knowledge of eccDNA biology. There is currently increasing concern about the connection between eccDNA and cancer. Gene amplification on eccDNAs is prevalent in cancer. Moreover, eccDNAs commonly harbor oncogenes or drug resistance genes, hence providing a growth or survival advantage to cancer cells. eccDNAs play an important role in tumor heterogeneity and evolution, facilitating tumor adaptation to challenging circumstances. In addition, eccDNAs have recently been identified as cell-free DNAs in circulating system. The altered level of eccDNAs is observed in cancer patients relative to healthy controls. Particularly, eccDNAs are associated with cancer progression and poor outcomes. Thus, eccDNAs could be useful as novel biomarkers for the diagnosis and prognosis of cancer. In this review, we summarize current knowledge regarding the formation, characteristics and biological importance of eccDNAs, with a focus on the molecular mechanisms associated with their roles in cancer progression. We also discuss their potential applications in the detection and treatment of cancer. A better understanding of the functional role of eccDNAs in cancer would facilitate the comprehensive analysis of molecular mechanisms involved in cancer pathogenesis. Extrachromosomal circular DNAs (eccDNAs), a major form of extrachromosomal DNAs, are widely present in various eukaryotic species, including drosophila The genome of eukaryotes consists of chromosomal DNA and extrachromosomal DNA elements which are physically excised from the chromosomes Increasing evidence indicates that eccDNAs in eukaryotic cells usually carry interspersed repeat sequences or tandemly repeated genomic sequences Hotta et al. in vitro using Xenopus egg extracts and sperm nuclei/naked DNA carrying telomere repeats in vitro was energy-independent and required residual amount of Mg2+. Altogether, these results suggest that eccDNAs could be produced from the chromosomes mediated by recombination-dependent and -independent mechanisms. Loss- and gain-of-functional analyses in appropriate cell/animal models may be conducive to verifying the importance of DNA replication in the process of eccDNA biogenesis. The detailed process of eccDNA generation is still elusive. So far, four potential models for eccDNA formation have been proposed, including the translocation-deletion-amplification model, the chromothripsis model, the breakage-fusion-bridge (BFB) model and the episome model  l Figure .MYC and AT motif binding factor 1 (ATBF1) was achieved through this model In the translocation-deletion-amplification model Figure A, translMYC was reported to be amplified via the chromothripsis model Chromothripsis is commonly found in cancer cells The BFB cycle proposed by McClintock is a well-established mechanism of genomic instability MYC-containing eccDNAs in leukemia, SCLC, and neuroblastoma cells Carroll et al. Additional mechanism has been proposed for eccDNA production. In lymphoblastoid cells exposed to chemotherapeutics, cell apoptosis could facilitate eccDNA production In recent years, eccDNAs have become a focal point of scientific research, and increasing reports have showed their involvement in a wide range of biological processes. It seems that different types of eccDNA possess distinct functions. Understanding the biological function of eccDNAs will lead to elucidating epigenetic mechanisms behind gene regulation under normal and pathological circumstances.mtDNAs play a crucial role in triggering innate immunity Figure . Collin Interestingly, mtDNAs can be encapsulated into extracellular vesicles (EVs) DAZ4, HLA and KIR, in human somatic tissue were prone to form circular products leaving chromosomal deletions Saccharomyces cerevisiae, the frequency of GAP1 eccDNAs and deletion of chromosomal GAP1 were increased under nitrogen-limiting conditions S. cerevisiae to nitrogen-limited environments. During the adaptive evolution in S. cerevisiae, xylose isomerase (XylA) gene amplification occurred through genesis of self-replicating eccDNAs S. cerevisiae. eccDNA formation followed by tandem gene amplification serves as a rapid means of adaptation to selective pressure during evolution. eccDNAs mediate gene translocation by integrating elsewhere in the genome. In cattle, a 492 kb chromosome 6 segment, BTA6, was circulated, reopened and integrated in the BTA29 on chromosome 29 BTA29 fragment, was translocated to the wild-type KIT locus within BTA6 by homologous recombination. These serial translocation events finally led to color sidedness in cattle.The eccDNAs can lead to deletions, mutations, replications, amplifications or translocation of genes, leading to intercellular genetic heterogeneity and adaptive evolution. Certain loci, including S. cerevisiae, two gene pairs, HTA1-HTB1 and HTA2-HTB2, encoded the histones H2A and H2B HTA2-HTB2 amplified via formation of eccDNAs under the condition in which HTA1-HTB1 was deleted. The eccDNAs, which carried HTA2-HTB2, the histone H3-H4 locus, a centromere and the origin of replication, were generated through recombination between two Ty1 retrotransposon elements that flanked this region. Therefore, eccDNAs serve a critical role in compensating for genetic loss.In eccDNAs have been associated with aging. The aging yeast was enriched for high copy eccDNAs Euglena gracilis. microDNAs harboring microRNA (miRNA) coding sequences were transcribed to form functional miRNAs that silenced their endogenous mRNA targets Oxytricha, abundant eccDNA molecules from nonrepetitive micronucleus (MIC)-limited sequences were generated during genome rearrangement Oxytricha. The exact mechanisms underlying the transcription of microDNAs remain to be clarified. In addition, microDNAs acted as protein sponges to combine with RNA polymerase subunits Interestingly, eccDNAs can be transcribed within cells, suggesting that they may have functional roles in cell physiology. Greenwood et al. Telomeric circles (t-circles) function as pivotal participants in telomere maintenance. In cancer cells, increased telomerase activity caused extended telomere length Increasing evidence has proven the existence of eccDNAs in circulation system Taken together, eccDNAs play a role in eliciting innate immune responses, mediating cell-to-cell communication, advancing genetic heterogeneity, compensating for genetic loss, aging, regulating gene expression and molecular sponging Figure . eccDNAsEGFR) gene were identified in patient-derived GBM cell lines. Koche et al. eccDNAs have been discovered in cancer tissues/cells by high-throughput sequencing. Recently, over 18,000 eccDNAs were identified in a pan-cancer analysis of ATAC-seq libraries from 23 cancer types, including glioblastoma multiform (GBM), lung adenocarcinoma (LUAD) and stomach adenocarcinoma (STAD) EGFR and c-MYC were found to be more amplified in human cancer tissues than normal tissues EGFR and MYCN) and their endogenous enhancers were maintained and amplified as extrachromosomal amplicons EGFR gene EGFR gene through the microhomology-based NHEJ pathway. eccDNA molecules including amplified MYC genes were universally present in human cancer cells MYCN gene was amplified as DMs in neuroblastoma and SCLC cell lines MYC-carrying DMs coexisted within the same leukemia cell population of patients with acute myeloid leukemia (AML) MYC gene was generated by a postreplicative excision from its original chromosomal location. The circularization of this amplicon might involve error-prone NHEJ pathway. It was likely that the scattered acentric DMs evolved toward ring chromosomes stabilized by neocentromeres, thus providing a selective advantage to leukemia cells.Increasing evidence has indicated that circular DNA molecules are important structures in oncogene amplification in cancer cells Compared with reports on ncRNAs, there are relatively few studies on eccDNAs. Therefore, it is necessary to comprehensively identify and characterize eccDNAs in cancer. Profiling the landscape of eccDNAs will provide important clues on the functional role of eccDNAs in carcinogenesis and cancer progression. Much more efforts should be made to compare the amount, sequence composition and topological characteristics of eccDNAs between cancer patients and normal controls. The screening of cancer-specific or -associated eccDNAs will lead to the discovery of novel biomarkers for cancer diagnostics and promising targets for anticancer therapeutics.eccDNA formation is an important mechanism responsible for the amplification of oncogenes Figure A. The macyclin D1 amplification in DMs and homogeneously staining regions (HSRs) was confirmed in primary bladder tumors cyclin D1 on DMs might be associated with clinical characteristics of patients. The oncogene Sei-1 was capable of inducing DM formation Met carried on DMs was significantly amplified and overexpressed. The Met signaling cascade in turn promoted the Sei-1-induced DM generation. High copy number amplification of eukaryotic initiation factor 5A2 (EIF5A2) was detected in ovarian cancer (OC) cells in the form of DMs EIF5A2 overexpression was significantly correlated with the advanced stage of OC. Downregulation of EIF5A2 by the reduction of DM copy numbers suppressed the growth of OC cells. Thus, EIF5A2 served a vital role in OC pathogenesis. Likewise, ribosomal L22-like1 (RPL22L1) was amplified via DMs in OC In vitro and in vivo evidence indicated that RPL22L1 could promote OC development. Gemcitabine was able to enhance the incorporation of DMs into micronuclei in OC cells EIF5A2, MYCN and myeloid cell leukemia-1 (MCL-1) present on DMs was decreased upon gemcitabine treatment. Accordingly, gemcitabine restrained the growth, colony formation and invasion of OC cells.The coexistence of NSMCE2, MYC and FGFR2) were identified to be amplified and overexpressed in human colorectal cancer (CRC) cells c-MYC could shift from an eccDNA to an intrachromosomal site. It is likely that integration of the oncogene into the chromosomal locus facilitates tumor progression.The universal occurrence of eccDNA-driven oncogene amplification was verified in glioblastoma MYC and lysine-specific methyltransferase 2A (KMT2A) were concurrently amplified on different DMs. These alternations may contribute to the pathogenesis of secondary myeloid malignancies. DMs in myeloid neoplasms generally contained MYC or mixed lineage leukemia (MLL) gene amplification and were linked with myelodysplasia or therapy-related disease Genetic aberrations were discovered in the leukemia cells of a breast cancer patient who developed secondary AML after chemotherapy There have been a large number of reports showing oncogene amplification in the form of DMs in cancer. Cancer cells harboring increased oncogene expression and copy number acquire a competitive advantage and possess the ability to rapidly fit changing environment. DMs play an important role in tumor heterogeneity, evolution and development. The mechanisms that drive oncogene amplification within DMs are worthy of further investigation. The critical factors that regulate the formation and amount of oncogene-carried DMs need to be determined. Furthermore, it is unknown whether the high-level amplification of oncogenes occurs within other kinds of eccDNAs. Thus, further research focusing on the oncogene amplification via other types of eccDNAs is required.DHFR, zinc finger FYVE-type containing 16 (ZFYVE16) and mutS homolog 3 (MSH3), and enhanced drug sensitivity in MTX-resistant cells. Similarly, suppression of NHEJ blocked DM generation, inhibited MTX resistance and cell proliferation in MTX-resistant colon cancer cells eccDNAs predominantly bear extrachromosomal amplification of drug resistance genes Figure B. The acEGFR gene and predominantly maintains on DMs EGFRvIII originates from a genomic deletion of the In human epidermoid carcinoma cells, the amplified multidrug resistance 1 (MDR1) genes were contained in DM molecules Extrachromosomal amplification of drug resistance genes serve as a vital mechanism responsible for cancer drug resistance. eccDNAs carrying drug resistance genes can drive intratumoral heterogeneity and contribute to extreme copy number amplification through their uneven segregation. Cancer cell-derived eccDNAs increase the diversity of genetic differences among offspring cells, thus conferring a proliferative or survival advantage to cancer cells under drug pressure. Suppression of eccDNA biogenesis may improve the therapeutic efficacy of anticancer drugs. More studies are needed to investigate eccDNA-mediated drug resistance in cancer, which will provide new insights into the epigenetic mechanism underlying cancer pathogenesis.mtDNA copy number variation has been correlated with cancer Reportedly, cancer patients had higher plasma concentrations of mtDNA compared with healthy controls It was reported that high mtDNA copy number in peripheral blood of cancer patients predicted a poor cancer prognosis while increased mtDNA copy number in tumor tissues was remarkably correlated with better overall survival in cancer patients The amount and nature of eccDNAs in cancer cells differ from that of normal cells In eukaryotes, the cellular landscape has different genetic components, such as chromosomal nuclear DNAs, coding RNAs, ncRNAs and so on. Besides chromosomal nuclear DNAs, eccDNAs are universally present in the nucleus and cytoplasmic organelles (e.g. mitochondria) of normal and malignant cells. In the past decades, extensive efforts have been made to uncover the mechanisms of eccDNA biogenesis. eccDNA formation may involve DNA replication-dependent or -independent processes. eccDNAs can be derived from either repetitive or nonrepetitive chromosomal sequences. Although several models have been raised to describe the biogenesis of eccDNAs, the precise mechanism and direct proof for these models deserve further research. It is still unclear how the segments of linear chromosomes circularize. More efforts are needed to characterize the topological structure and sequence composition of eccDNAs. Little is known about the mechanisms leading to the variation of copy numbers of eccDNAs. Additional work is required to figure out whether eccDNAs have the ability to self-replicate. At present, there is no consensus standard for eccDNA analysis. Different approach used in eccDNA study may have a marked influence on analytical results. Thus, it is important to standardize the procedure of eccDNA enrichment and data analysis. Further research is necessary to detect the basis of eccDNA formation across diverse species. eccDNAs have been studied for their role in genomic plasticity, instability, evolution, mutation and carcinogenesis. However, the functional roles of eccDNAs are yet to be fully delineated. Multiple studies have focused on the gene amplification capabilities of large-sized DMs. However, the role of microDNAs in gene amplification remains unclear. The gene amplification capabilities of smaller eccDNAs should be confirmed. Furthermore, there is a lack of direct evidence supporting the contribution of smaller eccDNAs to chromosome instability. These unanswered questions warrant further investigation to strengthen our knowledge regarding eccDNA function. The turnover of eccDNAs in cells or bodily fluids needs to be studied, which will provide new insights into their functions. Since cells produce diverse eccDNAs with heterogeneous origins, the connection between eccDNAs and different physiological/pathological states of normal cells is an intriguing area of ongoing research. Existing evidence indicates that a fraction of eccDNAs reside in the nucleus and can be transcribed. eccDNAs are likely to act as active gene transcripts and gene expression modulators. Thus, it is essential to define the impact of these eccDNAs and their gene products on the phenotype of cells.eccDNAs play a crucial role in cancer progression. eccDNAs confer an ability to amplify oncogenes and drug resistance genes. The proteins implicated in producing eccDNAs from the chromosome may serve as therapeutic targets for cancer. Therapies targeting eccDNAs may contribute to overcoming drug resistance in cancer. Nevertheless, the regulatory roles of eccDNAs in cancer pathogenesis remain to be verified. It is obscure whether eccDNAs directly regulate cancer cell proliferation, invasion and metastasis. Loss-of-functional analysis may be helpful to elucidate the effects of eccDNA depletion on cancer development. A comprehensive landscape of eccDNAs in neuroblastoma was previously profiled KLF17, CDH1 and LASS2) by decoying miR-9 that targeted these genes. The transfection of CSSD-9 via nanoparticles suppressed the proliferation and metastasis and favored the apoptosis of malignant cancer cells in vitro and in vivo. These results suggested that CSSD-9 might be used as therapeutic miRNA inhibitors to upregulate tumor suppressor genes and reduce tumor malignancy. Circular DNAs may be effective for sequestering miRNAs and provide a new perspective for the intervention of cancer. From a therapeutic viewpoint, an ideal ncRNA interference technology should display high stability and ncRNA inhibitory activity. eccDNAs may carry the favorable feature of resistance against nuclease degradation. Nevertheless, it remains to verify whether natural eccDNAs can act as ncRNA sponges. Specific sequences involved in the interaction between eccDNAs and ncRNAs are required to be identified. The feasibility of eccDNAs toward the regulation of tumor suppressor genes or oncogenes in vitro and in vivo models warrants further exploration. In addition, substantial studies should be carried out to evaluate the effectiveness and safety of eccDNA-based anticancer therapeutics. There are several difficulties that must be addressed going forward. One of the challenges is the risk of immune stimulation. The eccDNAs may be recognized by the innate immune system of target cells. Introduction of a large quantity of eccDNAs may induce non-specific interactions owing to the activation of innate immunity. It is important to design eccDNAs which can act at a low concentration. Additionally, the utilization of efficient delivery systems may ensure that eccDNAs can be applied at the lowest concentration. The off-target effect is another challenge with eccDNA-based therapies. The off-target effect can cause the suppression of ncRNAs that should not be targeted, which may lead to deleterious consequences. The screening and design of eccDNAs must be carefully performed to avoid this issue. eccDNA-based therapeutics that allow long-lasting and steady ncRNA-trapping at a low concentration may be feasible in enhancing therapeutic efficacy with a low risk of the off-target effect.Notably, circular DNAs hold tremendous promise as therapeutic ncRNA antagonists. Meng et al. Increasing evidence has demonstrated the potential utility of eccDNAs as a source of novel biomarkers for cancer detection and prognosis. Since eccDNAs can be detected in peripheral blood from cancer patients, cell-free eccDNAs may represent minimally invasive biomarkers for cancer. Despite the benefits of eccDNAs as potential novel biomarkers, there are several obstacles to be overcome. First, there are still many undescribed eccDNAs in cancer cells. Additional studies should be performed to screen and identify eccDNA candidates. It is believed that more and more eccDNAs will be discovered and characterized due to recent technological developments such as super-resolution microscopic method and next-generation sequencing. The difference in eccDNA contents between cancer patients and healthy controls must be ascertained. Deregulated eccDNAs may be useful in distinguishing cancer patients from healthy controls. Secondly, the approaches for eccDNA isolation, enrichment and quantification should be defined to ensure the accuracy of analytical results. Thirdly, the potential of eccDNAs as effective biomarkers is worthy of intensive investigation. Although many studies have indicated the altered amount of eccDNAs in cancer, the diagnostic and prognostic values of eccDNAs are elusive. Therefore, more clinical studies are necessary to assess the diagnostic/prognostic performance of eccDNAs in cancer.In conclusion, eccDNA study is still in its infancy, and the biological role of eccDNAs in cancer pathogenesis is just starting to be illuminated. There are many areas to address in this field, such as the biogenetic process of eccDNAs and the influence of eccDNAs on cell physiology. Increased research on the functional role of eccDNAs in cancer will broaden the horizons in the field of cancer biology and open up new opportunities for cancer diagnosis and therapy.