The coronavirus disease 2019 (COVID-19) crisis confronted us, like many researchers worldwide, with an unforeseen challenge during the final stages of a randomized controlled trial involving ataxia patients. Institutional guidelines suddenly no longer allowed regular follow-up visits to take place, impeding the clinical evaluation of long-term outcomes. Here, we discuss the various scenarios that we considered in response to these imposed restrictions and share our experience of home video recording by dedicated, extensively instructed family members. Albeit somewhat unconventional at first glance, this last resort strategy enabled us to reliably assess the study’s primary endpoint at the predefined point in time and hopefully encourages researchers in other ongoing ataxia trials to continue their activities. Remote assessments of ataxia severity may serve as a reasonable substitute in interventional trials beyond the current exceptional situation generated by the COVID-19 pandemic, but will require further investigation. The current coronavirus pandemic profoundly impacts patient care, medical education, and scientific research activities. Adaptation, flexibility, creativity, and problem-solving skills are highly required and more necessary than ever before. At the same time, institutional, national, and international guidelines must be followed accurately in order to prevent further transmission of the virus. Like many ongoing studies, our randomized, double-blind, sham-controlled SCA3-tDCS trial—in which we examine the effects of cerebellar transcranial direct current stimulation (tDCS) in twenty patients with spinocerebellar ataxia type 3 (SCA3)—is affected by the health security measures dictated by the authorities and hospital board . We hereWhen the COVID-19 crisis struck the Netherlands, our study had fortunately reached its final stage. All participants had completed the 10-day regimen of daily tDCS sessions and follow-up visit after two weeks to evaluate whether modulation of cerebellar excitability is able to reduce ataxia severity and a number of non-motor symptoms. Similar to most ataxia trials, absolute change in Scale for the Assessment and Rating of Ataxia (SARA) score from baseline was chosen as the primary outcome measure . FurtherThe statisticians ran a simulation study incorporating different scenarios and trajectories with four missing data points at twelve months follow-up but without actually using the real data. The results showed relatively limited influence of missing data on the estimated treatment effect, even in more extreme scenarios in which missing would primarily occur in one of the treatment groups. The statisticians thus argued against the completion of live assessments since the impact was deemed not large enough to justify the additional burden and risk.Inspired by other telemedicine initiatives at our outpatient clinic and elsewhere , we explhome), remote recordings of a SARA variant (iSARA) and of real-life gait through body-worn sensors, and Kinect-based movement tracking of SARA-inspired items in children at home (SaraHome) [Extraordinary times call for creative solutions that nonetheless could have important implications, not only for ongoing studies but also for future investigations. If we are able to perform interventional trials with reliable remote SARA assessments to evaluate changes in ataxia severity, this will probably benefit recruitment and retention and make studies more accessible to disabled patients. We are aware of ongoing developments in this area, such as app-based collection of home videos of patients performing a selected set of SARA items (SARAaraHome) . We are


Advances in medical diagnosis and treatment have facilitated the emergence of precision medicine. In contrast, locomotor rehabilitation for individuals with acquired neuromotor injuries remains limited by the dearth of (i) diagnostic approaches that can identify the specific neuromuscular, biomechanical, and clinical deficits underlying impaired locomotion and (ii) evidence-based, targeted treatments. In particular, impaired propulsion by the paretic limb is a major contributor to walking-related disability after stroke; however, few interventions have been able to target deficits in propulsion effectively and in a manner that reduces walking disability. Indeed, the weakness and impaired control that is characteristic of post-stroke hemiparesis leads to heterogeneous deficits that impair paretic propulsion and contribute to a slow, metabolically-expensive, and unstable gait. Current rehabilitation paradigms emphasize the rapid attainment of walking independence, not the restoration of normal propulsion function. Although walking independence is an important goal for stroke survivors, independence achieved via compensatory strategies may prevent the recovery of propulsion needed for the fast, economical, and stable gait that is characteristic of healthy bipedal locomotion. We posit that post-stroke rehabilitation should aim to promote independent walking, in part, through the acquisition of enhanced propulsion. In this expert review, we present the biomechanical and functional consequences of post-stroke propulsion deficits, review advances in our understanding of the nature of post-stroke propulsion impairment, and discuss emerging diagnostic and treatment approaches that have the potential to facilitate new rehabilitation paradigms targeting propulsion restoration. THE fast, economical, and stable gait that is characteristic of healthy bipedal locomotion –6 requirConventional post-stroke rehabilitation efforts have had limited effectiveness in restoring the propulsion function inherent to a healthy bipedal gait, with functional improvements often being the product of compensatory mechanisms , 30, 31.In this expert review, we discuss recent advances in our understanding of post-stroke propulsion deficits, review emerging approaches to systematically diagnose and treat the underlying impairments, and highlight the substantial research and development effort that is required before these approaches can alter clinical practice. More specifically, the next section on “Stroke Recovery and Rehabilitation Roundtable recalls that the “most recent phase III and IV trials have been largely neutral or negative”, citing “considerable urgency” for “ensur[ing] that our trials are mechanistically well conceptualized, with careful matching of the nature of the intervention and the outcome measure chosen”. The international group went on to identify a core set of clinical outcomes to be assessed in every stroke recovery trial, but also acknowledged that clinical outcomes alone are unable to distinguish between restorative and compensatory recovery strategies. Their final general recommendation was that “trials need to consider serially applied kinematic/kinetic measurements alongside clinical assessments to distinguish between restitution and compensation”. The group highlighted the importance of measurements that reflect the “quality of hemiplegic gait performance” and the role that technology will play in making routine clinical assessment of kinematic and kinetic measurements feasible.In their 2017 consensus statement , the StrThe incorporation of force measurements in the analysis of human locomotion was first enabled in 1938 with the development of a mechanical force-reactive platform by Herbert Elftman . Today, Despite the importance of propulsion to functional bipedal walking, the clinical management of impaired post-stroke propulsion is untenable without clinically-accessible approaches to characterize the extent and nature of paretic propulsion deficits. The lab-based tools traditionally used to assess propulsion  are not accessible to most clinicians. These tools are also not viable for measuring propulsion function in real-world settings where people live and move. Although experienced clinicians may be able to grossly estimate a person’s propulsion function based on visual observation of gait kinematics , such exWearable sensors have rapidly gained popularity for rehabilitative applications . These pSymmetrical interlimb propulsion during walking requires the normal functioning of multiple circuits along the neuromotor axis. Stroke induces a cascade of neurophysiologic changes in cortical and spinal circuits that either directly or indirectly disrupt the corticospinal tract, the principal pathway for the control of voluntary, fractionated movements –69. The Restorative therapies that aim to restore the normal functioning of the neural circuits affected by stroke may have strong potential to improve post-stroke propulsion. For example, neuromodulatory treatments such as repetitive TMS and transcranial direct current stimulation can be paired with gait training interventions to augment the excitability of the lesioned corticospinal pathways –84. PairDescending pathways emanating from non-lesioned cortical and subcortical circuits may show compensatory modulation of activity after stroke, and investigation of the role that neuromodulatory interventions can play in their function is warranted. For example, disrupted descending modulation of spinal circuitry may adversely affect post-stroke motor function. Indeed, increased excitability of spinal segmental reflexes, measured using H-reflexes, has been correlated with spasticity and excessive muscle coactivation after stroke , 89. SimThe individualization of propulsion-targeting gait therapies requires identification of the nature of the propulsion deficit and must thus account for the fact that both limb kinematics and kinetics affect propulsion , 103. InThe heterogeneity of propulsion impairment is present at still deeper levels of analysis. The plantarflexor muscles are the primary generators of propulsive force during healthy walking, and post-stroke plantarflexor weakness may be the result of a reduced strength capacity , reduced central neural drive, or a combination of these deficits. A promising diagnostic approach to elucidate the extent and nature of post-stroke muscle weakness combines dynamometry with supramaximal electrostimulation –107 Figa. For exBecause individual patients may have a combination of reduced plantarflexor central drive and strength capacity that contributes to their propulsion impairment, assessing each of these potential deficits may be necessary to inform clinical decisions. However, the diagnostic systems currently used for neuromuscular function testing require substantial time to setup and execute, as well as costly and large equipment not widely available in clinical settings. Together, these factors motivate the development of novel point-of-care plantarflexor force measurement systems that can integrate neuromuscular electrical stimulation to assess the extent of and mechanisms underlying plantarflexor weakness.A complementary assessment approach has recently emerged that leverages a posterior restraining force during walking to functionally assess an individual’s latent propulsion capacity, computed as the difference between an individual’s propulsion during unrestrained walking and their capacity to generate propulsion in the face of a restraining force—i.e., their “propulsion reserve” . While pIt has been suggested that features of post-stroke propulsion can be used to identify post-stroke gait phenotypes with different motor control deficits and that these phenotypes can be used to guide intervention matching , 112. OnCrucially, individuals with low Pp present with motor control profiles that are different from individuals with high Pp. Indeed, the gait of individuals with high Pp is characterized by increased EMG activity of the extensor muscles during terminal stance, a shortened paretic step length, and prolonged paretic hip extension . In contAnother approach to identify gait subtypes uses the peak paretic propulsion force (instead of the propulsion impulse) and combines this metric with a person’s walking speed to characterize individuals based on the combined knowledge of their walking performance  and quality . The co-assessment of these metrics was found to be substantially better at predicting the therapeutic response to a propulsion-targeting gait training program than either metric alone . There mTask-specific rehabilitation that emphasizes the direct practice of walking has emerged as a preferred approach to impairment-based training , 118; hoIn the face of persisting neuromotor deficits, passive walking aides are commonly prescribed to enable safe and independent walking. These devices, however, do not address deficits in paretic propulsion. Rigid ankle-foot orthoses (AFOs), for example, reduce drop foot during the paretic limb’s swing phase by constraining the ankle to a neutral position. By preventing plantarflexion during swing, the AFO enables safe ground clearance and reduces the risk of a fall; however, the other major role of a rigid AFO is to provide stability during the stance phase. In this role, the AFO may also limit plantarflexion during push-off, thus unavoidably limiting propulsion , 121. Ulfunctionally restore paretic propulsion deficits [In the last decade, technological and clinical advances have led to the development of novel rehabilitation programs and assistive devices that target post-stroke propulsion deficits. These have ranged from neuromodulatory interventions that facilitate activation of the impaired plantarflexors , 127 to deficits , 132.how walking speed and functional performance gains were achieved by this intervention approach as the large trials did not collect kinetic and kinematic data. In a smaller sample study (n=15) of this rehabilitation approach that assessed all individuals with stroke as a single cohort, improvements in walking speed were observed without concomitant increases in propulsion symmetry [et al additionally compared individuals who achieved clinically important improvements in walking speed  and individuals who achieved minimal gains  to determine which factors were associated with changes in walking speed [In the past 20 years, gait therapies that aimed to improve post-stroke walking function through repetitive stepping practice on a treadmill with body weight support and manual assistance, as needed, emerged as viable locomotor rehabilitation techniques . Howeversymmetry . Bowden ng speed . In thisng speed . A substng speed . Strikin–Traditionally, functional electrical stimulation (FES) is applied to the dorsiflexors to reduce drop-foot and facilitate ground clearance by the paretic limb. A novel training approach that applies FES to the plantarflexor muscles in combination with fast treadmill walking has emerged to target paretic propulsion deficits during walking Fig. , Left. T136Interestingly, despite only the FastFES group presenting with a substantial reduction in the energy cost of walking, between-group differences in 6-minute walk test distance were not observed  . Future in parallel with the underlying paretic muscles. Exoskeletons are rigid, brace-like structures that include on- or off-board actuation triggered and controlled by various sensors related to muscle function, joint kinematics, limb kinetics, and/or gait speed. Due to their rigid structure, they are capable of providing passive stability in non-actuated planes of motion. Recent advancements have allowed the creation of small and lightweight systems that can fit unobtrusively under or over clothes [In contrast to FES, rigid exoskeletons and soft robotic exosuits have been developed to generate assistive torques  clothes . Most ri clothes ,132, pre clothes  and gait clothes ,132. It TM soft exosuit   Fig. , Left reA) Fig. –, Right aDespite the impaired state of the paretic plantarflexor muscles, they often retain a remarkable force-generating capacity . This laThe approaches described above are intended to enhance gait by assisting patients to generate propulsion. The underlying assumption is that the individual does not have the capacity to increase propulsion on their own, requiring some means of external support . In contrast, there is growing evidence that individuals with chronic hemiparesis can be made to access a latent propulsive reserve . In partThe application of aiding and impeding forces to the body’s center of mass (COM) has a clear influence on propulsive forces and metabolic cost , 156. AiReal-time biofeedback of propulsion function is a promising intervention approach to exploit the presence of a propulsion reserve in neurologically-intact , 158–160The extensive prior work showing a strong relationship between post-stroke propulsion and walking ability , 129, co


In many ecosystems, plants represent the center of biological interactions. As the Earth entered the Cenozoic era, gymnosperm, and fern populations declined while flowering angiosperms became the dominant plant species. Plant pollination and seed dispersal mechanisms then evolved and diversified as animals began to feed on plant nectars and fruits. It is believed that these interactions drove the evolutions and diversities of both plants and other organisms. Even though many plant interspecific interactions have been studied, the molecular mechanisms regulating these interactions have not been characterized extensively. In many cases, plants secret various metabolites into the environment to act as signaling molecules detected by other organisms. Conversely, interactions with other organisms may also cause modifications in plant development, such as the generation of novel cell types and organs . Attractants are thought to consist of unique or unusual compounds that may explain parasite/pathogen's host range. However, it appears that most plant parasites and pathogens are in fact attracted to common compounds such as plant metabolites and/or plant hormones. Therefore, it seems the identities of attractants alone is not sufficient to explain plant parasite/pathogen's host range. Instead, the specific interactions between the host-secreted attractants and the parasite receptors may help to better understand how these interactions drove the evolution of both plants and their interacting partners .The first step of biological interaction between plants and other organisms, particularly parasites and pathogens, likely involves chemotaxis. Molecules secreted by plants often can act as guidance cues. Many sugars, organic acids, phenolics, amines, and phytohormones secreted by plants were shown to possess attractive properties to various animals and microorganisms, and some of their cognate receptors have also been identified . PPN have been documented to inject molecules, named effectors, to induce the differentiation of feeding cells , and to suppress the host plant's immune response . These effectors, associate with specific host proteins, enabling them to hijack important processes for cell morphogenesis and physiology and/or immunity. Some PPN-resistant plants have evolved intracellular nucleotide-binding domain leucine-rich repeat (NLR)-type receptors, which would recognize PPN effectors and induce NLR-triggered immunity .After parasites infiltrate their plant hosts, they may modify the host's signaling pathways to aid their own infection. Plant-parasitic nematodes (PPN), which include cyst nematodes and gall-inducing root-knot nematodes, are both scourges for agriculture and interesting case studies for such host manipulations. These nematodes spend the majority of their life cycles in plant roots, where they induce the formation of specific feeding structures inhabiting specialized feeding cells. These feeding cells known as giant cells and syncytia are multinucleate, hypertrophied, and hypermetabolic . Meanwhile, plant signaling pathways upon insect herbivory, a cascade of events including phosphorylation of a subset of transcription factors  by calcium-dependent protein kinases (CRK2 and CRK3), have also recently been characterized in Arabidopsis . These phytohormone-responsive CRKs may thus play major roles in the coordination of plant defense responses during insect herbivory. On the other hand, many social aphids are known to form elaborate galls on aerial plant organs, where up to thousands of individuals may occupy for up to over a year. Insect-induced galls are unique organs that provide both shelter and nutrients to plant-parasitic insects. In addition, aphids were also found to utilize the gall vasculature to remove wastes from the colony , suggesting the utilities of insect galls are indeed more complex than expected. Transcriptomic analysis of Chinese sumac (Rhus javanica) horned galls induced by aphids  revealed that meristem, flower, and fruit development master regulators as well as biotic and abiotic stress-responsive genes were highly upregulated in aphid galls .Aside from nematodes, insects are the other major multicellular animal taxon that interact with plants. The feeding of plant tissues from within plants (endophagy) is common among insects. Nutrition appears to be the main driver for the evolution of endophagy, however competition reduction, water conservation, and predation avoidance may have played significant roles , develop disc-like structures known as holdfasts in response to light and tactile cues, which are used to adhere to host plants (Shimizu and Aoki). Most parasitic plants then develop the root-like haustoria from the inner cortex, and invade the apoplastic space of the host's root. Once the haustorium reaches the host's vascular tissues, the parasite connects its own vasculature to the host's to acquire nutrients . Transcriptomic analyses using an in vitro Cuscuta campestris haustorium induction system revealed that genes involved in vascular stem cell development and proliferation were up-regulated in the haustoria in the absence of host, whereas genes required for xylem vessel cell differentiation were up-regulated only after the haustoria made contact with the host xylem. These results suggest host-derived signals and physical contact with the host vasculature are likely required to initiate haustoria xylem differentiation through transcriptional regulation .Not all plant parasites are animals. Limitations in soil fertility have influenced the diversification of nutrient acquisition strategies in plants, driven certain species to parasitize on other plants . Interestingly, these molecules are typically known to be involved in anti-microbial immune responses, yet here they have evolved to accommodate rhizobia colonization by escaping the host plant's innate immunity . Furthermore, two plant growth-promoting rhizobacteria (PGPR) strains, Bacillus sp. (12D6) and Enterobacter sp. (16i) were shown to be capable of rapidly colonizing the rhizosphere . Interestingly, inoculation of these PGPR strains to wheat (Triticum aestivum) and maize (Zea mays) rhizospheres significantly delays the onset of drought symptoms, which are likely due to root system architecture modifications induced by the PGPR .Plant interspecific interactions are not limited to herbivory and parasitism. One of the best-characterized symbiotic plant interspecific interactions is formed between leguminous plants and nitrogen-fixing rhizobia bacteria. These interactions take place in root nodules, which are unique root organs formed specifically to house the rhizobia. The host plants receive organic nitrogen from the rhizobia, in exchange for providing the rhizobia with carbohydrates as nutrients and nodules as accommodation. This mutualistic interaction has evolved sophisticated signaling networks that regulate rhizobia recognition, colonization, differentiation, and nodule formation. Cysteine-rich peptides, reactive oxygen/nitrogen species and toxin–antitoxin modules have all been documented to contribute to the regulation of legume-rhizobia symbiosis (The diverse collection of articles in this Research Topic highlights the vibrant and rapidly-changing research in the field of plant developmental modification during biotic interactions . These nAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Septic cardiomyopathy (SCM) is a complication that is sepsis-associated cardiovascular failure. In the last few decades, there is progress in diagnosis and treatment despite the lack of consistent diagnostic criteria. According to current studies, several hypotheses about pathogenic mechanisms have been revealed to elucidate the pathophysiological characteristics of SCM. The objective of this manuscript is to review literature from the past 5 years to provide an overview of current knowledge on pathogenesis, diagnosis and treatment in SCM. Sepsis is defined as a life-threatening organ dysfunction due to a dysregulated immune response to an infection  and has The myocardial depressant factors (MDFs) have been proposed since the 1950s , referriSome progress has been made in understanding the pathogenesis of SCM. In recent years, there are excellent review articles regarding the pathophysiology of cardiomyopathy, present review focuses on the current the evidence on pathogenic mechanism of SCM and the potential of diagnoses and treatments for SCM.Presently, the pathogenesis of SCM is still in its exploration stage. The role of inflammatory cytokines in the pathogenesis of sepsis was identified. But all antibodies based on specific pro-inflammatory cytokines as targets have failed in clinical trials . This inSepsis can lead to the activation of the complement system, resulting in increasing the amount of complement component 5 (C5a). C5a reacts with its receptor, leading to cytokine storm, lymphocyte apoptosis, neutrophilic innate immune function loss, cardiomyopathy, disseminated intravascular coagulation, etc. Meanwhile, C5a also affects intracellular calcium homeostasis . SCM is Cardiomyocytes have a high mitochondrial density, which allows them to produce adenosine triphosphate (ATP) quickly. ATP provides the energy for both energy-consuming endergonic reactions and energy-releasing exergonic reactions. Mitochondrial dysfunction can seriously affect heart function in sepsis. Mitochondrial damage occurs in SCM, mainly manifested as morphological and functional changes, including interrupted oxidative phosphorylation, impaired mitochondrial respiration rate, free radical production of mitochondria, decreased mitochondrial membrane potential, attenuated autophagy, and apoptosis . Takasu In summary, the findings from experimental animal models have shown that changes in mitochondrial morphology and function are involved in the pathogenesis of SCM. Understanding molecular mechanisms of mitochondrial injury may provide evidence for developing new therapeutic targets for SCM.2+ leakage in the sarcoplasmic reticulum (SR) of cardiac myocytes (Toll-like receptors (TLRs), a trans-membrane glycoprotein on the surface of the cell membrane, is an important part of the immune system that can identify different pathogen associated molecular patterns (PAMPs). The stimulation of TLRs by PAMPs causes the nuclear translocation of nuclear factor kappa B (NF-κB), and then leads to the expression of inflammatory mediators, such as TNF-α and interleukins (ILs) . Signalimyocytes . Chen etmyocytes . Based o2+ and also damage to mitochondria, etc. (Nitric oxide is synthesized by the oxidation of L-arginine by nitric oxide synthase (NOS) expressed in cardiac myocytes. NOS can be divided into three subtypes, namely neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) . The onlia, etc. . We haveia, etc. . This evAlpha 7 nicotinic acetylcholine receptor (α7nAchR) is widely expressed in the cytokine-producing immune cells such as macrophages, dendritic cells and T cells . α7nAchRin vitro and inflammatory response in vivo via the inhibition of exosome generation. In addition, the attenuation of cardiac dysfunction and improvement of survival are noted in septic mice  and ventricular dilatation during sepsis . HoweverTo date, there is no gold diagnostic standard for SCM. Most clinical studies use LVEF < 50% as a diagnostic criteria . Based oEchocardiogram: Echocardiography and doppler imaging can detect abnormalities in systolic and diastolic function of the heart in patients with severe sepsis or septic shock. There are three main indicators, namely myocardial performance index (MPI), mitral ring systolic displacement (MAPSE) and global longitudinal strain (GLS). MPI, also known as Tei index, is a parameter measured by echocardiography . The forModified shock index (MSI) refers to heart rate/mean arterial pressure, which can reflect both systolic and diastolic functions. Some biological markers, such as B-type natriuretic peptide (BNP) and troponin I are elevated in patients with SCM, which have no diagnostic value specifically . Chen etFor the diagnosis of SCM, cardiac dysfunction caused by other diseases should be excluded. It is important to distinguish it from acute coronary syndrome. Coronary angiography and coronary computed tomography angiography (CCTA) can determine whether there is myocardial ischemia. But it is a risky examination for the patients with sepsis. Some studies have suggested using ultrasonic myocardial perfusion technology to examine myocardial perfusion abnormalities in the area of impaired myocardial wall in patients with sepsis. SCM and coronary syndromes can be distinguished by this method, but it is still invasive . Also, oDue to the uncertainty of pathogenesis of SCM, no effective disease-modifying treatment is currently available. The strategies to deal with SCM is to control the primary disease to prevent the occurrence of secondary SCM. Specific treatment for SCM is still being developed. Currently some drugs and device-based therapy have been used clinically.Levosimendan is a calcium ion sensitizer, which can directly bind to troponin to increase myocardial contractility, but there is no significant change in heart rate and myocardial oxygen consumption. Studies have shown that levosimendan can effectively reduce the plasma lactic acid in sepsis and increase the myocardial contractility, but its application cannot reduce usage of norepinephrine and has no significant improvement in the mortality of SCM .Natriuretic peptide, a recombinant human brain natriuretic peptide (rhBNP), can bind to human endogenous natriuretic peptide receptor. Natriuretic peptide not only can expand the arteries and veins, urinate and expel sodium to reduce the preload and afterload of the heart, but also can inhibit renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system, which has been widely used clinically for treating heart failure via effectively relieving hemodynamic abnormalities and cardiac dysfunction. Combination of Chinese and western medicine: Clinical studies have shown the application of anti-infection and organ support therapy (MOST) accompanied by traditional Chinese medicine (fu zheng jie du hua yu granules) can further reduce acute physiology and chronic health evaluation (APACHE-II) score and other indicators such as procalcitonin (PCT), BNP and cTnI, in patients with SCM . Animal In septic patients complicated with SCM, extracorporeal membrane oxygenation (ECMO) is a feasible rescue strategy. Some approaches have been reported that application of anti-infection combined with ECMO has been used to treat the patient with SCM and septic shock caused by infection and hemorrhage of ileal diverticulum . Vogel eIntra-aortic balloon counter-pulsation (IABP) supports cardiac function by unloading contractions and enhancing relaxation. The former can lower systolic blood pressure and the latter can raise diastolic blood pressure. A retrospective study was performed among 38 cases with the application of IABP, suggesting that IABP is effective for the patients with cardiac shock by increasing mean arterial pressure and reducing dosage of catecholamine during the acute phase of sepsis. However, no improvement in long-term survival was observed . KenshirBlood purification has long been used to treat sepsis. Recently, Andreja Sinkovic reported that a patient with lymphoma, splenectomy and autologous bone marrow transplant and receiving chemotherapy, and accompanying severe pneumococcal infections, septic shock, SCM and unacceptable drug therapy, was subjected to blood purification. The reductions in the level of IL-6, lactic acid deposition and the dosage of vascular vasopressors, improvement of left ventricular systolic function and clinical features were observed after treatment for 36 h . CytoSorSome studies suggested that the pathogenesis of sepsis is the damage of micro-vessels caused by “Genomic Storm”. BacteriStructural and functional disorders of mitochondria affect the production of energy in cardiac myocytes, resulting in cardiac dysfunction. Maintaining the stability of mitochondrial structure and protecting its function have become the target of treating septic myocardium. Melatonin can restore the physiological functions of mitochondria and endoplasmic reticulum, maintain the stability of cytoskeleton, and thus improve cardiac function in septic mice . The stuIndependent growth factor I (GFI-I) can inhibit the expression of NF-κB and TNF-α, thus inhibiting LPS-induced inflammatory response and apoptosis of HPC2 cells . 3,3′-DiThe treatments discussed in this section are only from animal experiments and have not been applied clinically. Whether or not the genetic response in animal models can mimic human inflammatory disease is controversial.Septic cardiomyopathy, although it is reversible at early stage, has a high mortality rate because its pathogenesis is not well-understood. In terms of diagnosis and treatment, it is an important subject in clinical and basic research. Previous studies in this area have been limited by poor diagnostic strategies that only relied on LVEF reduction. In this review, we have not only discussed pathogenesis of SCM in detail, but also introduced some other approaches that are associated with the diagnosis of SCM. Early detection and intervention of SCM in patients with sepsis can reduce mortality. For example, MSI is considered as a “predictor” of SCM , and MAPHL and WW conceived and wrote the manuscript. ML, QM, and HR revised the manuscript. All authors listed wrote the manuscript and approved for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Flaviviridae family of RNA viruses includes numerous human disease-causing pathogens that largely are increasing in prevalence due to continual climate change, rising population sizes and improved ease of global travel. Escalating circulation of these emerging and re-emerging pathogens draws attention to the need for vaccines to protect against the severe diseases they cause, and this need is further exacerbated by their transmission that occurs primarily through arthropod vectors. When constructing new, efficacious vaccine candidates, several goals are targeted including safety, protective capacity, ability to confer sustained protection, induction of neutralizing antibody and protective T cell responses, as well as practicality.Prophylactic vaccination as a means to protect against flavivirus infection has become an intense area of research in the last several decades due to the inherent nature of this family of viruses to cause explosive outbreaks . HoweverAlthough the members of the flavivirus family have been shown to be antigenically similar , these vAlthough there is a currently approved DENV vaccine, safety and efficacy data support the need for alternative prophylactic vaccines to protect a broader population of individuals. Strides toward this goal include virus-like particles (VLPs), which are outlined in the review written by Wong et al. which summarizes VLP-based DENV vaccines currently being tested. As noted by Wong et al., many of the current VLP-based DENV vaccines lack the viral pre-Membrane (prM) protein; thus, it is believed these vaccines will circumvent the induction of cross-reactive non-neutralizing antibodies that may mediate antibody dependent enhancement. Importantly, these constructs still maintain key structural components that induce T cell and antibody responses, like envelope domain III (E DIII) against which most neutralizing antibodies are targeted . AdditioO-methylation enables mimicry of cellular mRNAs has been instrumental in reverse genetics studies to improve DENV vaccine design [Further, deeper understanding and advances in flavivirus genome structure and replication cycles have been able to aid the development of other kinds of unique DENV vaccine candidates. In this Issue, Park et al. outlines a candidate subunit vaccine based on eliciting a strong neutralizing antibody response against E DIII. This vaccine was developed to combat all four DENV serotypes by combining two subunits: partial envelope domain II and consensus envelope domain III (cEDIII) . The aute design . Specifie design ; this sae design .In addition to the previously mentioned challenges of developing flavivirus vaccines, another strong impediment to their development are practical considerations. As endemic regions of flavivirus circulation tend to be rural, access to trained medical professionals and refrigeration is often sparse. To counteract these obstacles, some vaccine constructs are aimed at achieving formulations that are biologically stable over time and do not require a medical professional to administer. In Muller et al., a tetravalent E DENV subunit vaccine was administered via a microarray patch and, when compared to traditional vaccination methods, led to enhanced neutralizing antibody titers to all four DENV serotypes potentially due to the delivery of antigens in close proximity to antigen presenting cells located beneath the skin. Inclusion of an adjuvant to enhance immunogenicity led to complete protection in mice following DENV challenge . In addiThis Special Issue also focuses its attention on Zika virus (ZIKV), a re-emerging pathogen with a history of causing epidemics: 2007 (Yap islands), 2013 (French Polynesia) and 2015 (South America) ,17,18. IIn addition to the need for development and maintenance of strongly neutralizing antibodies following vaccination, cellular responses have also proven to be essential in combatting ZIKV, as it has been shown that CD4 and CD8+ T cells are critical in reducing viral load and mortality in mouse models of ZIKV infection ,21,22. TAnother article, co-authored by our group, also focused on ZIKV vaccine development, highlighting another promising vaccine design avenue: adenovirus-vectors. These vectors have increased in popularity over the last several years as vaccine platforms primarily due to excellent safety profiles, the ability to be grown to high titers in cell culture, capacity to induce strong inflammatory immune response, etc. . SteffenAedes aegypti salivary gland protein, AgBR1, shapes innate immune responses in murine models following mosquito bites [As this Special Issue highlights, developing vaccines for numerous members of the Flaviviridae family has proven to be challenging for a variety of reasons, including virus interplay with the immune system, a concept highlighted in the review written by Tremblay et al. . One vacto bites . Based oto bites . This paHand in hand with vaccine design, methods of evaluation for vaccine efficacy are also under intense research. In a study by Frumence et al., a flow cytometry-based neutralization test (FNT) was employed as an alternative to the conventional plaque-reduction neutralization test (PRNT) as a means to enable a high throughput screening strategy that does not rely on methods requiring cell lines capable of fostering plaque formation. Through the development of a GFP reporter of ZIKV, this study highlights the ability to measure ZIKV neutralizing antibodies via flow cytometry with the antibody titers detected being equivalent to those done by PRNTs in tandem . AnotherOverall, this Special Issue highlights numerous studies, reviews and a case report that outline current research being done to further flavivirus vaccine development. These studies address both the challenges of developing flavivirus vaccines and the diverse and novel approaches to vaccine development that are currently underway. In addition, this Special Issue also features work being done to improve pre-existing vaccines, as well as techniques to streamline the process of determining vaccine efficacy while confirming safety. While the rates of flavivirus infections continue to rise globally, research efforts like the ones outlined in this Issue continually increase our knowledge of flaviviruses, thus informing the development of novel vaccines which will meet the growing need for protection against these pathogens.


In placental mammals, reproductive success, and maternal-fetal health substantially depend on a well-being placenta, the interface between the fetus and the mother. Disorders in placental cells are tightly associated with adverse pregnancy outcomes including preeclampsia (PE), fetal growth restriction, etc. MicroRNAs (miRNAs) represent small non-coding RNAs that regulate post-transcriptional gene expression and are integral to a wide range of healthy or diseased cellular proceedings. Numerous miRNAs have been detected in human placenta and increasing evidence is revealing their important roles in regulating placental cell behaviors. Recent studies indicate that placenta-derived miRNAs can be released to the maternal circulation via encapsulating into the exosomes, and they potentially target various maternal cells to provide a hormone-like means of intercellular communication between the mother and the fetus. These placental exosome miRNAs are attracting more and more attention due to their differential expression in pregnant complications, which may provide novel biomarkers for prediction of the diseases. In this review, we briefly summarize the current knowledge and the perspectives of the placenta-derived miRNAs, especially the exosomal transfer of placental miRNAs and their pathophysiological relevance to PE. The possible exosomal-miRNA-targeted strategies for diagnosis, prognosis or therapy of PE are highlighted. The placenta is a transient organ that plays a central role in maternal and fetal health during pregnancy . ServingMicroRNAs (miRNAs) are endogenous, small non-coding single-stranded RNAs, on average 22nt in length, which can regulate gene expression primarily through post-transcriptional repression or messenger RNA degradation in a sequence-specific manner . Most miin vitro and in vivo studies have revealed the vital roles of these placenta-derived miRNAs in the regulation of trophoblast cell behaviors and the occurrence of PE . The C19MC is the largest cluster of miRNAs in the human genome, and contains 46 highly homologous miRNA genes within a ∼100 kb genomic region . The miRExosomes are small extracellular vesicles of endocytic origin . They caExosomes contain multifaceted cargoes, including proteins, lipids, DNAs, mRNAs, miRNAs, LncRNAs, tRNA, and tRNA associated fragments . The sel1)In vivo and in vitro studies have demonstrated the dramatical repression of PRKG1 expression in maternal NK cells by exosomal miR-517-3p In vivo and in vitro results indicate the roles of placental exosomal miR-15a-5p in suppressing trophoblast cell proliferation, invasion, and apoptosis through downregulating CDK1 expression and hampering PI3K/AKT signaling, which is closely associated with PE progression ex vivo or in vivo evidence that supports the transfer of placental exosomes from the placenta to the fetus remains largely lacking.The placenta-derived exosomal miRNAs may regulate fetal vasculogenesis and angiogenesis. A study from 4)in vivo models are needed to clarify the working mechanisms of exosome transfer from the fetus to the placenta.A potential mode of exosomal miRNAs-mediated fetus-to-placenta signaling has been suggested. For instance, miR-133b in human umbilical cord mesenchymal stem cell (MSC)-derived exosomes boosts trophoblast cell proliferation, migration, and invasion via targeting SGK1 gene . ExosomaPreeclampsia has long been the leading cause of maternal and fetal morbidity and mortality, affecting approximately 2–7% of pregnancies. It is defined as the sudden onset of hypertension after the 20th week of gestation in pregnant women who had no preexisting hypertension, accompanied by significant proteinuria or multi-system symptoms, such as pulmonary oedema, seizures, or oliguria . A well-in vivo and in vitro results have demonstrated the participation of these aberrantly expressed miRNAs in PE-associated placental defects (Great efforts have been put to identify genes or signaling pathways that are associated with the deregulation of trophoblast differentiation and the development of PE . A great defects .For PE, a big challenge is the discrepancy between the key pathophysiological changes that are initiated well before the 20th week of gestation and the clinical symptoms that are not manifested until after that. Therefore, a critical thought is whether these differential placental miRNAs really contribute to the pathological change of PE or they are just the consequences of the disorder at late gestation . As statCirculating miRNAs can be encapsulated into extracellular vesicles including exosomes or bound to stabilizing proteins (mainly Ago proteins; It has to be noticed that the results from various studies may show different changing patterns of the exosomal miRNAs in PE patients , 2019. TIn recent years, RNA-based medicine is receiving growing attention for its diverse roles and promising therapeutic capacity . InteresThe discovery of placenta-derived miRNAs and their multiple roles in maintaining healthy pregnancy undoubtedly represent one of the most exciting progresses in recent years. In addition to the canonical intracellular silencing functions, placenta-derived miRNAs can also be released to the maternal or fetal circulation via encapsulating into the exosomes, and therefore potentially target various recipient cells to provide a non-hormonal means of intercellular communication between the mother and the fetus. Furthermore, unique exosomal miRNA profiling is potential diagnostic or predictive and prognostic tool for pregnant complications such as PE, and provides novel treatment targets for the disease.in vivo and ex vivo models should be constructed to address this point, which is indispensable for developing exosomal miRNA-based therapeutic strategies for pregnant complications such as PE. Finally, the convenient and controllable detection of exosomal miRNA remains challenging, because the adequately simple and robust assay platforms are lacking. A recent wok by There exist several interesting topics that require further investigations. First, studies regarding exosomal miRNA in pregnant women have been predominantly focusing on the total exosomal miRNAs, while seldom identifying their diverse origins. A recent report reveals that the origin of exosomes determines its target cells and the transfer activity , indicatPX and YM drafted the manuscript. HW participated in reference mining. Y-LW designed and supervised the study, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therapeutic nucleic acids hold immense potential in combating undruggable, gene-based diseases owing to their high programmability and relative ease of synthesis. While the delivery of this class of therapeutics has successfully entered the clinical setting, extrahepatic targeting, endosomal escape efficiency, and subcellular localization remain as major roadblocks. On the other hand, viruses serve as natural carriers of nucleic acids and have acquired a plethora of structures and mechanisms that confer remarkable transfection efficiency. Thus, understanding the structure and mechanism of viruses can guide the design of synthetic nucleic acid vectors. This review revisits relevant structural and mechanistic features of viruses as design considerations for efficient nucleic acid delivery systems. This article explores how viral ligand display and a metastable structure are central to the molecular mechanisms of attachment, entry, and viral genome release. For comparison, accounted for are details on the design and intracellular fate of existing nucleic acid carriers and nanostructures that share similar and essential features to viruses. The review, thus, highlights unifying themes of viruses and nucleic acid delivery systems such as genome protection, target specificity, and controlled release. Sophisticated viral mechanisms that are yet to be exploited in oligonucleotide delivery are also identified as they could further the development of next-generation nonviral nucleic acid vectors. Undruggable targets are disease-implicated proteins that lack easy-to-bind pockets where conventional therapeutics like small molecules can bind . Howeverin vivo delivery. This is particularly challenging given the limited systemic stability of unmodified nucleic acids. Thus, an ideal delivery strategy should include nucleic acid protection from nuclease degradation and oxidation, prolonged systemic circulation, targeted delivery, efficient transfection across a membrane, facilitated access to the cytoplasm or nucleus, and little to no side effects  and neuraminidase (NA), matrix proteins (M1 and M2), the heterotrimeric RNA-dependent RNA polymerase (RdRP), the viral RNA segments, a nucleoprotein (NP), and two nonstructural proteins (NS1 and NS2 a.k.a. nuclear export protein or NEP). The outermost layer of the virus is a lipid membrane decorated with glycoproteins that, in turn, may be recognized by antibodies to protect the host against infection . Thus, t in vivo . As per Another example is the adenovirus (AdV), one of the largest (90–100 nm) non-enveloped double stranded linear DNA viruses. The icosahedral shaped capsid is made of many structural polypeptides. Most of the capsid coat (about 75%) is composed of a hexon protein, which is held together by protein IX. A unique feature of Adv capsid is that the vertices are made of a penton protein from which fiber knobs protrude out–both of which are essential for host cell entry. The viral genome is condensed by proteins V, VII and μ and is also covalently associated with the terminal protein. The cementing protein IIIa acts as capsid stabilizing protein by linking the facets of the icosahedron . AdenoviDespite the structural and mechanistic differences among viruses, all viral capsids are metastable, which means they are stable enough to protect the genome until they reach the target site to uncoat it. Thus, the virus construct is spring-loaded in that potential energy is stored during its assembly. Upon reaching the target site, a chemical trigger such as low pH or proteolytic enzymes overcome the energetic barrier, resulting in virus disassembly and uncoating of the genome. Metastability is achieved by the inherent symmetrical arrangement of identical capsid protein subunits that is stabilized by nonspecific noncovalent interactions. In this regard, many capsid proteins self-assemble into virus-like particles (VLPs) .VLPs are non-infectious, multiprotein complexes that mimic the viral capsid assembly but are devoid of the genome. Their utility as experimental tools and as therapeutic carriers has been thoroughly reviewed elsewhere . RecombiLike viruses, VLPs have been successfully used in developing vaccines and vaccine adjuvants, and their use in gene therapy and immunotherapy has also been explored . Some of4 crosslinker, the VLPs were chemically labeled with m-lycotoxin, a cell-penetrating peptide, to enhance cellular uptake.Target specificity can be tailored by chemical conjugation or directly expressing targeting ligands on the protein coat . For exaWhile the ability of viruses and VLPs to efficiently encapsulate and transfect nucleic acids is remarkable, they are structurally more complex and, thus, typically require hosts for production and subsequent purification , both ofUtility of Inorganic Nanoparticles) form an electrostatic interaction with the negative phosphate backbone of the nucleic acid cargo, providing protection from nuclease degradation . pDMAEA can then be copolymerized with a second block of p(N-(3-(1H-imidazol-1-yl)propyl)acrylamide (pImPAA) and poly(butyl acrylate) (pBA) that mediates an acid-triggered endosomal escape. PImPAA and PBA were designed based on viral membranolytic peptides, and they disrupt the endosomal membrane synergistically through electrostatic and hydrophobic interactions, respectively . Such caNucleic acid protection through charge neutralization and condensation by cationic materials may only provide partial nuclease resistance . MoreoveAmong these, liposomes have shown the most promise . They arModular release usually centers on the lipid formulation where the lipid envelope is destabilized either by an external stimulus such as temperature or an cellular stimulus such as low pH . As an eOn the other hand, pH-sensitive liposomes exploit the differential acidification in the vicinity of malignant tumors or within endosomes for controlled release via membrane fusion or destabilization . Earlier®), a liposomal vector developed by Alnylam Pharmaceuticals, as the first US Food and Drug Administration approved synthetic carrier of siRNA into cells  . These a3O4) nanoparticle, when utilized as a delivery vehicle, can be stimulated to produce oscillating magnetic fields which could then promote more efficient endocytosis . SNAs radially display a high density of nucleic acids around a spherical nanoparticle. The introduction of high concentrations of salt masks the polyanionic backbone of the nucleic acids, permitting clustering around a very small surface area . MoreoveThe overall 3D architecture of spherical nucleic acids (SNAs) imparts nuclease resistance through steric-shielding and enhanced local ionic strength . This stDesigned to build upon the successful properties of SNAs, NADNs utilize densely packed oligonucleotides around a scaffold, enhancing oligonucleotide stability and permitting scavenger-mediated endocytosis but are built upon biodegradable core materials. The scaffolds of reported NADNs are chemically diverse  and can Cellular Targeting, Attachment, and Entry), endosomal escape (Section Cytosolic Delivery), and nuclear delivery (Section Nuclear Delivery).Overall, this section underscores that virus particles are metastable machines built to protect the viral genome and that its overall responsiveness to the environment enables it to carry out its function as an infectious particle. In a similar fashion, nonviral synthetic carriers are designed to protect nucleic acid cargo and facilitate controlled release. Tropism is the ability of viruses to target specific cell types by binding their surface protein or peptide ligands to specific host cell receptors. The elaborate means with which they make use of these ligands accounts for their cell target specificity and high uptake efficiency . MechaniPrior to entry, viruses often adhere to the cell surface via non-specific electrostatic interactions involving viral surface components (i.e. membrane glycoproteins) and negatively charged sugars (i.e. heparin sulfate) attached on the target cell surface . Though vβ3 on the cell surface  that are normally inaccessible to the therapeutic . Many no surface . The suc surface . An RGD- surface .Other ligands of non-viral origin also offer targeting properties. For example, monoclonal antibodies have a been highly effective at targeting delivery of cytotoxic drugs to cancer cells . Their aDs in the pico-to nano-molar range) (Nucleic acid aptamers offer another promising approach in delivering nucleic acid cargos to specific cell-types . Aptamerr range) . Althougr range) . For exar range) .Lastly, small molecules are commonly used as targeting ligands as they are easily synthesized at a modest cost. They are more stable than biological ligands such as aptamers and peptides, and their conjugation is often relatively simple. However, these molecules are often not the natural ligands of the target cell receptors and thus have lower affinity and specificity for a given receptor, the latter giving rise to off-target effects. Nevertheless, the relative structural simplicity and functional designability of small molecules make them attractive and viable targeting domains .D = 1 nM) and minimal toxicity. Folate-functionalized vectors are typically internalized via receptor-mediated endocytosis, but reduced folate carriers, though having lower affinity, directly enter the cytosol. Folate-expressing imaging agents are currently in Phase I and Phase II clinical trials, but they are not yet clinically approved for targeting therapeutic nanoparticles  is widely used for targeting folate receptor-positive cell lines, with a high affinity  target sigma receptors that are upregulated in cancer cell lines. Benzamide analogues can also target dopamine receptors selectively. So far, these have been used to deliver small molecule drugs such as doxorubicin encapsulated in liposomes but have not been explored in gene-delivery yet .2) with spatially concentrated sialic acid functionalities on the surface of the host cell (50–200 per 100 nm2) is necessary for effective attachment and uptake  was the first US Food and Drug Administration approved GalNAc conjugate for acute hepatic porphyria, and other conjugates are underway  for hepatic targeting. GalNAc involves multi-site interactions with asioglycoprotein receptors (ASPGR) of hepatocytes, facilitating endocytosis. . In 2019underway . ASPGR iunderway . The strAc units . SpecifiAc units  spaced fAc units .Other synthetic vectors having multivalent interactions with cell receptors have been developed to mimic viral behavior and have shown an enhanced cellular uptake of the carriers or nucleic cargo. A prime example of this is the study of + T cells. In particular, maraviroc binding induces a change to the inactive conformer of CCR5  peptide is a targeting and endosomal escape peptide that has been used in siRNA delivery . GALA waThe fact that fusogenic or membranolytic peptides are often required to gain cytosolic access underscores the necessity for an endosomal escape component in a drug delivery system. This idea has been extended to various small molecules that can be used as tools to cross the endo-lysosomal membrane either through direct conjugation to or co-delivery with the nucleic acid cargo . For exaAdditionally, For effective host cell infection, the Lassa virus LASV,  and ebolIn particular, LASV was found to bind mainly to α-dystroglycan  as well Similarly, attachment of EBOV to the host cell membrane facilitates internalization principally through macropinocytosis , with evBecause NPC1 is involved in vesicular trafficking, it is even more interesting that it is responsible for limiting lipid nanoparticle-mediated siRNA delivery by shuttling the bulk of the lipid nanoparticles back to the outside of the cell after endocytosis . MoreoveUnlike cytoplasmic viruses, nuclear viruses  need to travel further in order to replicate themselves in the nucleus of the host cell. They must cross a total of three cell barriers to reach the nucleus—the plasma membrane, cytosol and the nuclear membrane. Thus, they have evolved to use their structural features along with cellular transport machinery to hijack the well-protected nuclear import process. The size, structure, and composition of the viral proteins determines the mechanism by which it enters the nucleus. The structure and surface properties of nuclear viruses are also different from cytoplasmic viruses as the capsid of these viruses needs to be intact when they are traversing through the highly crowded cytosol but should breakdown in the perinuclear area .The nucleus is the main regulator of intracellular functions such as gene activation, cell division and proliferation, metabolism and protein production. As such, it is also considered as the most important target to deliver intact therapeutic exogenous oligonucleotides to treat diseases at the genetic level . HoweverTo reach the nucleus, a number of different cytosolic trafficking strategies have been explored by nuclear viruses. Among these, the karyopherin-dependent and microtubule-assisted pathways have been extensively studied and mimicked for nucleic acid delivery . Thus, tThe nuclear trafficking of the viral ribonucleoproteins (vRNPs) is required for production and release of mature virions. To travel actively toward the nucleus, viruses use nuclear localization signals (NLSs) to mediate nucleus entry of the vRNPS. NLS sequences are short basic peptide motifs that are recognized by karyopherin proteins and are transported to the nucleus via karyopherin α/β-mediated pathway . Detailein vivo. In addition, plasmids complexed with proteins like HMG-1, histone H2B proteins, karyopherin receptors, and nucleoplasmin show increased transgene expression due to nuclear uptake  is a 72 bp aptamer derived from SV40 and has innate affinity for NLS-tagged cytoplasmic proteins such as transcription factors (TFs) . DTS-conr uptake .Many viruses use microtubule (MT) facilitated transport to traverse the cytoplasmic medium. Viral proteins induce rearrangement of microfilaments and recruit molecular motors such as dynein and kinesin to traverse from the plus to the minus terminal of MTs . The MT-In an effort to mimic viruses, the dynein binding protein (DBP) is often used as a ligand for nuclear uptake as it can mediate the transport of cargo via the MT-assisted pathway . A revieEvolution has honed viruses to be master hijackers of a broad range of host cells. They possess unique structural and mechanistic features wherein overarching themes such as capsid metastability, genome protection, stimuli-responsiveness, receptor duality, and synergistic ligand activity make them attractive templates for the design of non-viral nucleic acid carriers. Based on these outstanding characteristics of viruses, it is evident that an ideal carrier needs to find a balance between nucleic acid protection and release, two seemingly contradictory functions. A dynamic structure that responds to site-specific cues such as low pH or enzymatic activity help to control the release of nucleic acid cargo. These cues can vary with microenvironments within a cell, enabling biochemically controlled release mechanisms. Alternatively, the vector can be made sensitive to external stimuli such as light or temperature, which is more applicable to locally delivered formulations .While therapeutic nucleic acids have made it to the clinical setting, extrahepatic targeting, endosomal escape, and controlled subcellular localization remain as major hurdles in their delivery . VirusesIn many ways, the outstanding difference in the transfection efficiency of viruses and synthetic vectors stems from the lack of a consensus of what drives endosomal escape. Escape from the endosome is influenced by a large range of factors such as nanoparticle properties , mode of cellular uptake, and the type of cell . MoreoveIn summary, viruses can serve as a source of inspiration for chemists and materials scientists alike in the design considerations of non-viral vectors due to their efficient uptake and delivery of nucleic acid cargo. By designing nanoscale materials with stimuli-responsive properties and efficient targeting and internalization, therapeutic nucleic acids can be more rapidly brought forward for clinical application.


Patients with a serious mental illness often use cannabis at higher rates than the general population and are also often diagnosed with cannabis use disorder. Clinical studies reveal a strong association between the psychoactive effects of cannabis and the symptoms of serious mental illnesses. Although some studies purport that cannabis may treat mental illnesses, others have highlighted the negative consequences of use for patients with a mental illness and for otherwise healthy users. As epidemiological and clinical studies are unable to directly infer causality or examine neurobiology through circuit manipulation, preclinical animal models remain a valuable resource for examining the causal effects of cannabis. This is especially true considering the diversity of constituents in the cannabis plant contributing to its effects. In this mini-review, we provide an updated perspective on the preclinical evidence of shared neurobiological mechanisms underpinning the dual diagnosis of cannabis use disorder and a serious mental illness. We present studies of cannabinoid exposure in otherwise healthy rodents, as well as rodent models of schizophrenia, depression, and bipolar disorder, and the resulting impact on electrophysiological indices of neural circuit activity. We propose a consolidated neural circuit-based understanding of the preclinical evidence to generate new hypotheses and identify novel therapeutic targets. Cannabis is one of the most widely used psychoactive substances worldwide, and patients with serious mental illnesses use cannabis at rates much greater than the general population , 2. The As described below, cannabinoids produce distinct changes in neural circuit electrophysiological activity that are similar to those observed in patients with serious mental illnesses, as well as rodent models of these illnesses , 44. NeuCannabis users inhale smoke or vapor from crudely burning cannabis flower or by vapourizing it at greater temperatures . ResearcSchizophrenia is a complex neuropsychiatric illness characterized by severe dysfunctions including delusions, hallucinations (and other “positive” symptoms), social withdrawal (and other “negative” symptoms), and deficits in memory and sensory processing (and other “cognitive” symptoms) . ConsideIndividuals diagnosed with major depressive disorder present with symptoms such as persistent negative affect, anhedonia, as well as disturbed sleep and appetite . In rodeThe symptoms of bipolar disorder are characterized by cyclic changes in mood, motivation, and attention, ranging from periods of manic to depressed symptoms . Althougin vivo LFPs from the prelimbic cortex (PrlC) and hippocampus (HIP) showed reduced theta power in the HIP and PrlC and low gamma (30–50 Hz) power only in the PrlC  before, or after, exposure to D-amphetamine (2 mg/kg) to produce an acute manic episode. CBD did not affect amphetamine-induced hyperlocomotion in this rat model, which contradicts existing literature and may reflect innate differences in Wistar Kyoto rats . The modUnderstanding the pathophysiology of co-occurring cannabis use and serious mental illnesses remains a challenge in neuropsychiatry. Aberrant theta, alpha, beta, and gamma oscillations are observed after cannabinoid exposure in humans and in mental illness, while cannabinoid exposure differentially impacts the symptoms of the afore-mentioned mental illnesses. Similarly, preclinical models exhibit suppressed baseline neural circuit oscillatory activity, while cannabinoid exposure produces differential effects on electrophysiological neural circuit activity in otherwise untreated animals and has mixed effects in preclinical models on oscillatory activity, neuroplasticity, and neuronal firing rates. That said, our mini-review  shows that aberrant gamma activity is consistently observed. Whether aberrant gamma is etiological or only a consequence remains contested; however, the rescuing effect of gamma modulation implies that it is involved in mediating pathophysiological mechanisms , 113.Gamma oscillations arise from competing excitatory and inhibitory control, involving extensive coordination between glutamatergic and GABAergic transmission, and the pathogenic loss of inhibitory control via impaired interneurons may contribute to aberrant gamma oscillations , 33, 114Although the review focused on schizophrenia, major depressive disorder, and bipolar disorder, it is important to consider which other serious mental illnesses may involve, or be precipitated or ameliorated by, cannabinoids and eCB modulation. Patients with attention-deficit/hyperactivity disorder, obsessive-compulsive disorder, anxiety-related disorders, and post-traumatic stress disorder all report greater rates of cannabis use and worsened outcomes , 118. SoFuture studies should focus on comparing the effects of varying administration routes, toward a set of standardized methodologies used to examine cannabinoid exposure in models of mental illness, as these studies often use different routes and doses of cannabinoid administration which may contribute to inter-study variability. The other cannabinoids in the cannabis plant must also be investigated, as they demonstrate partial agonist activity for both CB1R and CB2R, and produce signature CB1R-dependent behavioral responses in mice tested using the cannabis tetrad . AdditioBJ drafted the review and formatted The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bet-hedging is a complex evolutionary strategy involving morphological, eco-physiological, (epi)genetic and population dynamics aspects. We review these aspects in flowering plants and propose further research needed for this topic.Bet-hedging is an evolutionary strategy that reduces the temporal variance in fitness at the expense of a lowered arithmetic mean fitness. It has evolved in organisms subjected to variable cues from the external environment, be they abiotic or biotic stresses such as irregular rainfall or predation. In flowering plants, bet-hedging is exhibited by hundreds of species and is mainly exerted by reproductive organs, in particular seeds but also embryos and fruits. The main example of bet-hedging in angiosperms is diaspore heteromorphism in which the same individual produces different seed/fruit morphs in terms of morphology, dormancy, eco-physiology and/or tolerance to biotic and abiotic stresses in order to ‘hedge its bets’ in unpredictable environments. The objective of this review is to provide a comprehensive overview of the ecological, genetic, epigenetic and physiological aspects involved in shaping bet-hedging strategies, and how these can affect population dynamics. We identify several open research questions about bet-hedging strategies in plants: 1) understanding ecological trade-offs among different traits; 2) producing more comprehensive phylogenetic analyses to understand the diffusion and evolutionary implications of this strategy; 3) clarifying epigenetic mechanisms related to bet-hedging and plant responses to environmental cues; and 4) applying multi-omics approaches to study bet-hedging at different levels of detail. Clarifying those aspects of bet-hedging will deepen our understanding of this fascinating evolutionary strategy. Organisms must cope with a variety of threats to their survival, ranging from abiotic stresses, such as the lack of resources or unfavourable climatic conditions, to biotic stresses such as predation and infections. Plants, being sessile organisms, evolved different strategies to overcome these environmental factors, depending on their temporal variability. When environmental variation is consistent, such as seasonal regularity, physiological or developmental plasticity is sufficient to allow individuals to adapt their phenotypes in response to the prevailing conditions. When, on the contrary, environmental variation is unpredictable, organisms apply diverse options without predicting how it will affect their future fitness, thereby ‘hedging their bets’ , in the embryos or fruits, and occasionally also in vegetative tissues such as buds Fig. a Charle. It is pGiven the importance of bet-hedging in angiosperms, we review its ecological and evolutionary implications with a focus on seed dormancy and heteromorphism, but also considering special cases such as bud dormancy, apomixis and serotiny. Despite a conspicuous number of studies describing bet-hedging at the ecological level, especially investigating how morphology and germination patterns affect fitness of different plant populations and taxa, still little is known about the molecular mechanisms underlying this phenomenon. We start by providing a resume on the different definitions and categorizations of bet-hedging, moving to its diffusion in flowering plants and its consequences for the dynamics of population genetics. We then review the genetic bases of seed heteromorphism, citing the most recent works linking different loci to bet-hedging strategies. We also describe how transcription and its regulation can drive bet-hedging in plants, including via epigenetic mechanisms triggered by environmental cues. Finally, we provide information on the physiological aspects that are linked to bet-hedging, such as hormonal regulation and antioxidant responses, and we conclude by suggesting further research that is needed on this subject.In a stable environment with predictable fluctuations, the fitness of phenotypes that are specialized to those conditions is maximized and constant over time, and thus, variance of fitness is low or null. In the same environment, generalists are disadvantaged as, even if with low variance, their mean fitness is lower Fig. a. On theIn other words, bet-hedging is a trade-off between the mean and the variance of fitness  diversified, several specialized strategies at once but fixed, e.g. variable seed sizes drawn from a fixed distribution; (iii) ‘adaptive coin flipping’, e.g. randomly produced seed sizes; and (iv) combined, any combination of the above.Olofsson et al.  defined Diaspore heteromorphism, or the production by the same individual of two or more seed/fruit types, is considered one of the main bet-hedging strategies in flowering plants  type, two seed morphs are specialized for two different conditions (e.g. wet/dry years), thus maximizing the mean and the variance of fitness of both seeds. On the contrary, in the high-risk/low-risk (HRLR) heteromorphic species both seed types are specialized for the same conditions (e.g. wet years), but the fitness of one morph is higher in optimal conditions and lower in different conditions, thus increasing both its mean and variance of fitness (high risk) compared to the other morph (low risk), whose mean fitness is lower but less variable over time. Seed heteromorphism is common in soil seed banking, in which a fraction of seeds remains dormant after dispersal within the soil or on its surface (Imbert Venable  describee Imbert . A propoe Imbert .Picris echioides L. (Asteraceae) produces two types of achenes: the peripheral ones are dispersed by mammals and remain enclosed in the involucral bract, while the central achenes are wind-dispersed  Andrz. ex DC) , heteromorphic caryopses are produced within the same spike and variations in colour, mass, dormancy, abiotic stress tolerance, longevity and phenology can be observed among the different morphs, contributing to the adaptation to ruderal or disturbed environments  Pall. (Chenopodiaceae) differ in several traits that allow the adaptation of this species to saline soils, including different dormancy levels, seed coat thickness and colour, and resistance to saline stress  Freitag & Schütze variations in seed heteromorphism, in terms of plant size, seed number and heteromorphic seeds ratios, have been associated with maternal environmental factors  can be modelled to derive the threshold values that permit germination in a fraction of the seed population (Bradford Ψb(50), is lower and its standard deviation is higher. Indeed, larger water potential ranges allow larger differences in the germination fraction over different years or rain events within the same year, thus leading to plastic germination and bet-hedging strategies  and water potential (Bradford . These mMammillaria (Cactaceae): in M. pectinifera F.A.C. Weber seeds are expulsed actively during rainy periods and passively over years in order to spread the chances of seedling establishment over time  exerted by the environment Dyer . HoweverBet-hedging strategies are diffused in many plant families in different plant development phases and anatomical parts, but to the best of our knowledge, it has been phylogenetically quantified only in terms of seed heteromorphism and persistence within the soil seed bank. Lamont and colleagues 2020) describe the phylogenetic distribution of serotiny, present in eight angiosperm families; however, this distribution is not yet resolved at the species level. Heteromorphism was reported to be present in 218 plant species and is more frequent in dicotyledonous plants , habitats and seed traits such as dormancy. Persistent seed banks, suggesting bet-hedging strategies, are mostly diffused in weedy taxa with annual life cycles living in disturbed and ruderal habitats  redIn particular, it has been demonstrated that long-term soil seed banking seeds show an equal or higher nucleotide substitution rate when compared to less persistent seeds, and that the majority of seed bank-borne mutations are neutral or nearly neutral, in accordance with Otha and Kimura’s neutral theory of molecular evolution  (Soliman Primula vulgaris Hudson (Primulaceae) was explained, in part, by genes linked to flower morphology and the influence of the light and temperature environment experienced by the female parent during the flowering period (Vaerbak & Andersen Packera tomentosa (Asteraceae) to genetically differentiate clones (genets) that showed cryptic heteromorphism, i.e. a variable seed behaviour such as dormancy that is not accompanied by a discriminant morphological variation  Husn.) and its progenitor, wild emmer wheat (Triticum turgidum subsp. dicoccoides (Asch. & Graebn.) Thell.) (Poaceae), in order to characterize heteromorphism to clarify the domestication syndrome in wheat. While domesticated emmer shows uniform grain size and germination, wild emmer bears, within the same spikelet, bigger caryopses located in the upper part that germinate more rapidly when compared to their smaller siblings located in the lower part of the spikelet. A quantitative trait locus (QTL) for uniform grain size and germination on chromosome 4B (QGD-4BL) explained a high proportion of within-spikelet variation in terms of caryopses dimensions and dormancy, indicating a modification selected during early stages of domestication.  analysed the molecular bases of within-spikelet heteromorphic caryopses exploiting a recombinant inbred line (RIL) population originated from durum wheat , two loci, one overlapping with DELAY OF GERMINATION 6 (DOG6) and the other with DOG1  and FLOWERING LOCUS T (FT) enable A. thaliana mother plants to modulate seed dormancy in the progeny depending on the external temperature, sensed through the epigenetic state of these genes. This mechanism allows the modification of dormancy levels through the regulation of seed coat development and hormone production in response to environmental conditions during seed maturation to diversify the behaviour of the progeny, presumably to maximize fitness in the following generation , and subtle differences in the seed response contribute to bet-hedging via the formation of transient or persistent seed banks , genetically identical except for two loci involved in abscisic acid (ABA) catabolism (RABA1—Reduced ABA 1) and sensitivity (SOG1—Suppressor of gamma response 1), showed a bet-hedging strategy based on a continuum of ABA-dependent dormancy. Allelic differences at these loci were correlated with dormancy release when seeds were subjected to different temperatures, with the two lines showing different lower limits of tolerance . This rare tree species relies on a mixed reproductive strategy utilizing both sexual embryonic production and agamospermy gene from Pennisetum squamulatum (L.) R.Br. and BABY BOOM1 (BBM1) in rice (Oryza sativa L.) , fruit dimorphism (dehiscent or indehiscent) is a ‘last-minute’ developmental decision, happening after fertilization. In this species, flowers that already possess the structures typical of the dehiscent morph (four to six ovules and a segment) can degrade those structures and instead form indehiscent fruits , belonging to the family of transcription factors TB1 CYCLOIDEA PCF (TCP). BRC1 transcription is indeed particularly important in hormonal production and is thought to integrate the regulatory roles of developmental hormones such as auxin, strigonolactone and cytokinin that are involved in fruit development  and WRI1 (embryo development). These results, suggesting different degrees of after-ripening in the two morphs, are in accordance with the already known dormancy behaviour of the two morphs, while other DEGs  suggest also the possibility of different longevity behaviour, so far unknown  showed the establishment of a bet-hedging strategy possibly induced by the inheritance, within the same generation, of random epiallelic variants caused by the heat stress experienced by the ancestor , generating plastic phenotypic variation that can be heritable or not. Phenotypic plasticity can thus be individual (within-generation) or be inherited as an effect of the epigenetic changes exerted by the environment on the parents that is meiotically transmitted . The latter can be defined as ‘heritable bet-hedging’ and can maintain or increase the genetic potential of a population across generations, acting as a means of evolutionary rescue from extinction  and in the perennial herb Helleborus foetidus Moench (Ranunculaceae)  was related to within-plant variance in seed size  K.Koch and Arthrocnemum indicum (Willd.) Moq. (Chenopodiaceae), two halophytes with black/brown and large/small heteromorphic seeds, respectively, differential levels of antioxidant activity and oxidation markers  were found during seed germination under increasing salinity. In both species, the salt-tolerant morphs did not show changes in their antioxidant activity nor in H2O2 and MDA levels, while the morphs with lower salt tolerance also showed a less resilient antioxidant machinery, resulting in higher levels of oxidative damage  showed different lifespans and antioxidant activities: the shorter-lived, larger morphs possess lower antioxidant activities when compared to their smaller, darker and longer-lived counterparts  possesses two morphs, black and brown, in which there is differential accumulation of phenolics in the seed coat. The black morph, with slower water uptake, constitutes the soil seed bank, whereas the brown one is salt tolerant and shows a more rapid germination, due to its permeable coat and a higher GA content  Asch. & Graebn. (Fabaceae)) collected across the Mediterranean area, south-eastern Europe and the Middle East. The soluble to insoluble PAs ratio and coat thickness correlated with different dormancy levels in differently distributed populations, this in turn correlating with different seasonality and climatic conditions and thus indicating a bet-hedging strategy in wild peas , showing plasticity in dormancy release along with an aridity gradient. A genome-wide association study revealed that four genes related to flavonoid metabolism and seven peroxidases and thio-/peroxiredoxins are associated with differential dormancy release depending on the environmental conditions  due to the ‘collateral’ antioxidant activity of chemicals involved in dormancy regulation, e.g. phenols  can embody these strategies by exhibiting physical (e.g. seed size) or physiological (e.g. seed dormancy) heteromorphism . Bet-hedging is also reflected in differential transcription patterns of genes belonging to different metabolic and developmental processes, including embryo development, fatty acids and sugar metabolism, ROS detoxification and late embryogenesis abundant proteins-LEAs (Footitt et al. Different loci have been associated with seed heteromorphism and bet-hedging, mainly comprising genes involved in pathways linked to dormancy (e.g. Epigenetics can drive bet-hedging via trans- and inter-generational transmission by regulating gene expression through genome-wide methylation marks in response to environmental cues. The regulation of different pathways affects physiology through differential hormone levels, antioxidant responses and seed coat properties in heteromorphic seeds (Abley et al. Bet-hedging strategies result from ecological adaptions and are driven by different genetic, epigenetic and physiological processes that in turn modify the dynamics of population genetics Fig. :Bet-hedgMany aspects of bet-hedging in flowering plants still need to be explored. From an ecological point of view, most of the studies on bet-hedging have focused on just one phenotypic trait, such as seed longevity (Guzzon et al.


Fibrous dysplasia (FD) is a rare, sporadic, and benign congenital condition in which normal cancellous bone is replaced by fibro-osseous tissue with immature osteogenesis. FD localized in the cranial and facial bones is called craniofacial fibrous dysplasia (CFD). Cystic degeneration in CFD cases is rare; cystic degeneration appearing in both the maxilla and the mandible FD lesion is even rarer. The aim of this article was to report a case of fibrous dysplasia of the mandible and maxilla complicated by nonspecific cystic degeneration.A 30-year-old woman presented with a rare case of non-specific cystic degeneration in a mandible and maxilla FD lesion that occurred 11 years after surgery. She was diagnosed with polyostotic CFD and underwent maxillary and mandibular bone contouring. Cyst enucleation under general anesthesia was performed in the mandibular region due to pain and discomfort.In cases involving non-aggressive and non-invasive FD cystic degeneration in focal areas, conservative treatment is recommended. However, if cystic degeneration of FD develops rapidly and causes discomfort, pain, or dysfunction, surgical treatment should be considered. Fibrous dysplasia (FD) is a benign disorder characterized by the replacement of normal bone tissue with proliferative fibrous connective tissues . SomaticNon-epithelial-lined cysts sometimes occur in association with various benign and malignant bone lesions, including FD, giant cell tumors, chondroblastoma, ossifying fibroma, benign osteoblastoma, cemento-osseous dysplasia, fibrous histiocytoma, fibrosarcoma, and osteosarcoma. These cysts show various patterns and can appear as aneurysmal bone cysts, simple bone cysts, or non-specific cystic degenerations .Cystic degeneration in CFD cases is rare; cystic degeneration appearing in both the maxilla and the mandible is even rarer. A PubMed search from 1946–2019 (using the search terms “fibrous dysplasia” [Ti] OR “McCune–Albright” [Ti] OR “Jaffe–Lichtenstein” [Ti] OR “Mazabraud” [Ti] AND (cyst [Ti] OR cystic [Ti]) initially identified 78 articles. After screening and manual review, a total of seven articles on the occurrence of cystic degeneration in maxillary or mandibular FD were identified .In November 2018, a 30-year-old woman presented to the Department of Oral and Maxillofacial Surgery of Chonbuk National University Hospital with a complaint of pain and swelling in the left mandible that had appeared 10 days earlier. Eleven years ago, she was diagnosed with CFD  showed normal response from #34, 35, and 36. No evidence of gum inflammation, such as periodontal pockets or gingival sulcus swelling and bleeding, was found during the periodontal examination. Furthermore, in the panoramic view, the dental origin with the possible infection source was not observed  showed an expansive bone lesion with a ground-glass appearance spanning the left frontotemporal bone, crista galli, orbital wall, ethmoid bone, sphenoid bone, zygoma, pterygoid plate, and maxilla regions Fig. a. An illA decision to perform a marsupialization procedure was made to first control edema and pain, and second to take a biopsy. The marsupialization procedure was performed after an intraoral incisional biopsy of the area surrounding the #34 and #35 teeth, followed by root canal treatments on these teeth. The biopsy results revealed some evidence of chronic inflammation and that the lesion may be a bony lesion with inflammatory reaction rather than FD. During a 3-week observational period, the size of the lesion was unchanged according to clinical and radiological findings  stained sections showed dense collagenous tissue surrounding the osseous trabeculae, and peritrabecular clefting was present Fig. a. MitosiIn view of the radiographical and intraoperative findings, the absence of any history of trauma, a low probability of dental infection being the cause , absence of evidence of malignant transformation  –15, the The FD cysts with nonepithelial lining have different histological characteristics, in which some are aneurysmal bone cysts, whereas others are simple bone cysts or nonspecific cyst degenerations . The forCystic degeneration of CFD is most often found in the sphenoid and frontal bones of patients with FD. Pressure from the cysts on the optic nerves can cause acute optic nerve compression in patients with acute cystic degeneration (ACD) of CFD . Cysts tAccording to the National Institutes of Health cohort study (unpublished observations), cystic degeneration occurred in approximately 5% of all patients with FD , whereasCystic degeneration of FD may exhibit a rapid increase in size, which could be misdiagnosed as a malignant transformation. If FD shows a clear lytic appearance or a rapid increase in size, the possibility of cystic degeneration or malignant transformation should be considered. Cystic degeneration of FD that shows an aggressive pattern on radiological findings tends to have poorly defined borders, osteolytic changes, and erosion of the cortex with periosteal reaction. This pattern is similar to that of the malignant transformation of FD , making Surgical treatment for FD is limited to cases involving esthetic or functional problems. It is recommended when FD poses a threat to important anatomical structures, such as the eyes or the optic nerves, causes significant esthetic deformities, or if severe pain is clearly associated with the FD process . In someIn this case, our patient had a rare case of cystic degeneration simultaneously in the maxillary and mandibular FD. The mandibular lesion showed discomfort and pain, and cyst enucleation was performed. However, follow-up was decided on the maxillary lesions because of the absence of pain or discomfort in that region. We found no noticeable differences in maxillary lesions on CBCT at 6 months follow-up Fig. d. The poCystic degeneration in CFD cases is rare; cystic degeneration appearing in both the maxilla and mandible is even rarer. In this case, our patient had a rare case of nonspecific cystic degeneration simultaneously in the maxillary and mandibular FD. The mandibular lesion caused discomfort and pain, and cyst enucleation was performed. Continuous follow-up is required for long-term prediction of prognosis.


Extrachromosomal circular DNA was recently found to be particularly abundant in multiple human cancer cells, although its frequency varies among different tumor types. Elevated levels of extrachromosomal circular DNA have been considered an effective biomarker of cancer pathogenesis. Multiple reports have demonstrated that the amplification of oncogenes and therapeutic resistance genes located on extrachromosomal DNA is a frequent event that drives intratumoral genetic heterogeneity and provides a potential evolutionary advantage. This review highlights the current understanding of the extrachromosomal circular DNA present in the tissues and circulation of patients with advanced cancers and provides a detailed discussion of their substantial roles in tumor regulation. Confirming the presence of cancer-related extrachromosomal circular DNA would provide a putative testing strategy for the precision diagnosis and treatment of human malignancies in clinical practice. It is therefore important to explore the extrachromosomal DNA profiles of patients with advanced cancers in clinic Table . It has sitivity –136. Liksitivity , 138. Basitivity . Thus, nH-ras [K-ras [EGFR and androgen receptor (AR) into extracellular space, which enhances the malignant phenotypes of neighboring cells [In addition, it has recently been proven that some extracellular DNA is enriched in cancer-derived extracellular vesicles (EVs), further contributing to their stability . In humaH-ras  and K-ras [K-ras , and ress [K-ras , 145. A ng cells . Of courIn light of their important roles in oncogene plasticity and drug resistance, circular DNA elements can be detected in the blood-based screening of plasma from cancer patients as a component of liquid biopsy , 148. EsWhile circular extrachromosomal DNA has been able to be determined for some time, higher-resolution imaging and cytogenetic methods have only recently been developed to evaluate these molecules. Advances in sequencing technologies have allowed for the genome-scale screening and mapping of circular DNA from human cancer cells . Based oIt is also essential to expand the cytogenetic toolbox to explore the function of key components with respect to extrachromosomal DNA particles. For a long time, fluorescence in situ hybridization (FISH)-based approaches have been used to resolve amplified oncogenes linked with cancer development. However, FISH probes have been unable to discriminate the location of specific genes on chromosomal and extrachromosomal DNA molecules . A more EGFR and c-Myc) could help cancer cells adapt more effectively to variable environmental stress by acquiring fate-enhancing advantages. Thus, some subclones would express higher levels of oncogenes, leading to cancers that become more aggressive, have poorer prognoses, and are more difficult to treat clinically over time. Accordingly, understanding the underlying molecular mechanisms of tumor heterogeneity, including oncogene amplification from circular extrachromosomal DNA, might help to develop remarkable potential therapeutic strategies that either prevent cancer progression or overcome therapy resistance. Such important scientific topics are currently being addressed by pioneering novel work on these issues.As mentioned above, cancer-associated genes can be amplified in circular extrachromosomal DNA elements. In particular, because of the unequal segregation into daughter cells, the amplification of eccDNA/ecDNA elements could be a primary mechanism by which oncogenes rapidly reach higher transcript levels and copy numbers than in intrachromosomal amplification. There is growing evidence supporting the functional importance of circular DNA in promoting genetic heterogeneity and accelerating the progression of human cancer pathologies. Moreover, the amplification of circular DNA elements containing oncogenes (such as