The softening phenomenon of age-hardening aluminum alloy-welded joints is severe during conventional fusion welding, which increases the likelihood of stress and strain concentration in the joint during the period of service, significantly reduces the mechanical properties compared to the base metal, and represents an obstacle to the exploration of the potential structural performance. This review paper focuses on an overview of the softening phenomenon. Firstly, the welding softening mechanism and the characteristics of age-hardening aluminum alloys are clarified. Secondly, the current main research methods that can effectively improve joint softening are summarized into three categories: low-heat-input welding, externally assisted cooling during welding, and post-weld treatment. The strengthening mechanism and performance change rule of age-hardening aluminum alloy joints are systematically analyzed. Finally, this paper considers the future development trends of further research on joint softening, and it is expected that interest in this topic will increase. Age-hardening aluminum alloys are widely used in structural design in modern aerospace, automotive, and other transportation industries due to their high specific strength, good formability and corrosion resistance, which can meet the requirements of light weight under the premise of ensuring safety and reliability ,2,3. HowAccording to the strengthening mechanisms of aluminum alloys, they can be divided into two categories: (I) heat-treatable aluminum alloys and (II) non-heat-treatable aluminum alloys , as showAt present, the generally accepted precipitation sequence of the strengthening phase in age-hardening aluminum alloy is supersaturated solid solution→GP zone→coherent precipitate→semi-coherent precipitate→non-coherent precipitate, as shown in The age-hardening aluminum alloy in the peak-aged state has high strength owing to a large number of precipitates that are very fine and coherent, and contributes to the effective resistance to dislocation movement and can cause an intense strengthening effect ,14. Howe(1)The characteristics of the precipitation phase in BMsThe mechanical properties of age-hardening aluminum alloys highly depend on the strengthening phase, which precipitates at a specific temperature range ,35,36. TThe characteristics of the precipitation phase in BMs, which is determined by the temper and chemical composition, also have a great influence on the joint efficiency. Compared to peak-aged aluminum alloys, the underaged or overaged aluminum alloys have lower mechanical properties and their precipitation is less sensitive to the welding thermal cycle. Therefore, the joint efficiency of peak-aged aluminum alloys is lower than that of under-aged or over-aged aluminum alloys.(2)The thickness of welding platesFor the same temper and chemical compositions of age-hardening aluminum alloys, thicker plates need a larger welding heat input than thinner plates to achieve sufficient penetration ,38, whic(3)Welding technique and processThe welding method with low energy density and the welding process with large heat input would significantly reduce the joint efficiency due to more serious softening in FZ and HAZ.For aged-peak aluminum alloys, joints welded by conventional fusion welding can roughly be divided into five areas, namely FZ, HAZ1, HAZ2, HAZ3 and BM, that reflect the effect of different temperatures on the strengthening phases, as shown in It is obvious from the above-mentioned discussion that the larger the welding heat input, the greater the degree and range of age-hardening aluminum alloy joint softening. Moreover, in contrast to other welding defects, such as porosity and cracks, that can be eliminated by improving the welding process, softening is inevitable. Hence, improving the effect of welding heat on the microstructure and properties of age-hardening aluminum alloy joints has increasingly become the focus of researchers.According to the characteristics of softening in age-hardening aluminum alloy joints, researchers have adopted various effective technologies and processes to reduce the joint softening and improve the performance of welding joints, which can be summarized into three categories, as shown in (1)Low-heat-input welding: such as laser beam welding (LBW), friction stir welding (FSW), and cold metal transfer (CMT) welding. To improve the mechanical properties of age-hardening aluminum alloy joints welded by the low heat input technique, researchers have focused on how to eliminate welding defects ,42,43,44(2)Externally assisted cooling: involving spraying a cooling medium onto the surface of the workpiece and submerging the workpiece in the cooling medium. To reduce the effect of the welding thermal cycle on strengthening precipitates, researchers have sought to control the peak temperature range and enhance the cooling rate during welding ,55.(3)Post-weld treatment: including post-weld heat treatment, post-weld surface modification treatment and post-weld rolling treatment. To further improve the mechanical properties of age-hardening aluminum alloy joints, researchers have sought to strengthen the softening zones through post-weld treatment, causing over-aged strengthening phases to reprecipitate or introducing dislocation strengthening ,57,58.The precipitation strengthening mechanism of age-hardening aluminum alloys is easily affected by the welding heat input. The high heat input undoubtedly results in extensive over-aging of strengthening phases and increases the degree and range of joint softening, which seriously affects the mechanical properties of welded joints. Therefore, in recent years, welding research on age-hardening aluminum alloys has mainly focused on welding methods with low heat input characteristics, such as LBW, FSW and CMT. Although the mechanical properties of joints produced with low-heat-input welding methods have been improved, there are still some welding problems. Based on this, researchers have made great efforts to solve these problems, so that the mechanical properties of the joints can be further improved and the application scope of the low-heat-input welding method can be expanded.For age-hardening aluminum alloys using conventional fusion welding with a low energy density, excessive heat input often causes the weak position of the joint to be located in the FZ or HAZ; thus, the performance of the welded joint severely declines, and the joint efficiency can be reduced to less than 50% . HoweverCompared to conventional fusion welding, Compared to pure aluminum, the addition of alloying elements such as silicon, copper, magnesium and zinc to aluminum to generate the effect of precipitate strengthening by the thermal treatment process can increase the crack susceptibility in the FZ during autogenous laser welding . The higFor autogenous laser welding, the FZ contains a large number of cellular dendrites; only the center of the FZ is slightly equiaxed. The grain microstructure in the FZ is correlated with the solidification rate and temperature gradient. The solidification grain microstructure map created by Kurz and Fischer is presented in R is the solidification rate, V is the welding speed, and α is the angle between the welding direction and the normal surface of the solidification front, which is determined by the weld shape.During the welding process, an equiaxed crystal structure can easily form with a high solidification rate and low-temperature gradient, and cellular dendritic grains can easily form with a low solidification rate and high-temperature gradient. The local solidification rate is as expressed in the following equation :(1)R=VcoLaser welding with beam oscillation can adjust the solidification rate by controlling the weld shape, and thus improve the grain microstructure in the FZ. As shown in 6 W/cm2, while the value for aluminum alloys is at least 1.5 × 106 W/cm2 and even 2 × 106 W/cm2, because aluminum alloys possess high laser radiation reflection and thermal conductivity [When laser welding thick aluminum alloy structures, the keyhole-welding mode requiring a high-power laser source should be adopted to obtain high penetration depths. However, some critical issues usually arise during the welding process: (I) high energy consumption—the energy density (ratio of the laser power to the beam spot area) for iron-based materials in the keyhole-welding mode is approximately 10uctivity ,86; (II)uctivity ,88; Optimizing shielding gas environmentCai et al.  found th(2)Applying a magnetic or ultrasound field during the welding processLiu et al.  investig(3)Optimizing welding processHuang et al. ,104 repoIn the FSW process, due to the friction heat between the tool shoulder and the workpiece surface, the material enters into a plastic softening state. Under the effect of the rotating pressure of the pin, the softened material undergoes plastic flow, fills the cavity generated by the moving of the tool and forms the weld ,107,108,Although the heat input of FSW is lower than that of conventional fusion welding, the microstructure morphology and properties of welded joints are still changed under the influence of the welding thermal cycle ,140,141.The process parameters that can affect the joint performance of FSW aluminum alloys include the shape of the pin and shoulder , the tooAs the welding situation is fixed, only the rotating speed and welding speed directly determine the welding heat input during the friction stir welding process ,148, andMost studies have shown that ,140, witResearchers found that when the welding speed is too slow, the plastic metal accumulates on the backward side of the stirring pin, which leads to the formation of voids in the forward side due to the lack of sufficient metal filling . HoweverIt is well known that reducing the welding heat input can effectively decrease the softening of age-hardening aluminum alloy joints and improve the strength of the joint. However, many studies reported that the low heat input during the FSW can decrease the plastic flow capacity of the material and thus introduce welding defects, such as kissing bonds, voids, tunnels and “S” lines, which dramatically reduce the strength of the joint ,149. HenBlaha and Langenecker found that the use of ultrasonic vibration could reduce the yield stress and promote the plastic deformation of the metals , which wIn addition, Hu et al. ,51,152 sDuring the traditional MIG welding process, when the liquid bridge achieves contact with the molten pool, the electromagnetic shrinkage force acting on the liquid bridge is increased by instantly increasing the short-circuit current, which causes the successful metal droplet transition to the molten pool. The increase in short-circuit current will undoubtedly increase the welding heat input. The CMT technique was developed on the basis of the traditional MIG welding technique. However, when the liquid bridge achieves contact with the liquid molten pool, the CMT welding machine control system reduces the shorting-circuit current to almost zero, and the metal droplet transition to the molten pool is aided by the back-drawing force exerted by the wire feeding system , as showComparing CMT with other types of fusion welding, many researchers have confirmed that CMT has “colder” characteristics. The characteristics of CMT, with low heat input and less spatter, are highly suitable for welding age-hardening aluminum alloys that are easily affected by the welding temperature and cause serious deterioration in properties ,157,158.Although the conventional CMT technique is suitable for welding age-hardening aluminum alloys due to its low heat input, the thickness of base metals is limited to no greater than 3 mm, also due to the low energy input ,164. If (I)Hybrid CMT and another type of welding: Liang et al.  adopted (II)Control arc mode: Pang et al.  adopted The strengthening precipitates of age-hardening aluminum alloys are vulnerable to the peak temperature range and high-temperature residence time during welding. Therefore, the externally assisted cooling techniques, which reduce the peak temperature range and enhance the cooling rate during welding, can improve the mechanical properties of age-hardening aluminum alloy joints by controlling the effect of the welding thermal cycle on strengthening precipitates.FSW is a solid-state joining technique; although the temperature during welding does not cause the age-hardening aluminum alloys to melt, it is high enough to cause the dissolution and coarsening of strengthening precipitates in the joint , formingAccording to the action position of the cooling medium, the FSW with externally assisted cooling techniques can be summarized into two categories: (I) FSW with cooling medium on the surface of the workpiece, as shown in The cooling medium has a great influence on the mechanical properties of the joint. Fratini et al.  carried To improve the stability of the arc and control the welding defects in the process of fusion welding of age-hardening aluminum alloys, the externally assisted cooling mode of spraying a cooling medium onto the surface of the workpiece or submerging the workpiece in the cooling medium cannot be adopted. However, a high-thermal-diffusion welding pad can reduce the heat input of the joint and improve the influence of the welding heat cycle on the performance of the welded joint. Liang et al.  investigAlthough the joint softening of age-hardening aluminum alloys can be reduced by the low-heat-input welding methods and can be controlled by the externally assisted cooling technique, softening that occurs mainly due to the precipitation strengthening effect being weakened in the FZ and HAZ is inevitable after welding. The differences in the performance of the FZ and HAZ from that of the base metal result in stress–strain concentration during stress deformation , which rVarious effective measures have been developed and utilized by researchers to avoid and overcome softening when welding joints of age-hardening aluminum. However, currently, post-weld heat treatment (PWHT) is still considered the best method to improve the properties of the soft zone, especially for thick-plate joints. The main purpose of PWHT for age-hardening aluminum alloys is to cause the joints to reprecipitate the coherent precipitation phases, resulting in a homogeneous distribution of the fine strengthening precipitates and improving the mechanical properties of the joint back to the state of the base materials ,184. HowStress corrosion, fatigue properties, fracture properties and other failure behaviors of welded joints are often affected by the material state near the surface . Hence, 2) irradiated through a 1–2 mm confinement layer  onto an ablative layer  covered by the material surface produce high-temperature and high-pressure plasma, as shown in High-density laser pulses  gas passed through a Laval nozzle expands, which can accelerate the powder particles to a high velocity and violently impact the substrate, producing extensive deformations and metallurgical bonds ,204,205,Li et al. ,206,207 (1)Post-weld entire rolling: After welding, the entire plate  is rolled to a thickness less than the as-received base metal , as shown in Rolling, as an important process to increase the dislocation density and improve the strength of materials, is commonly used to strengthen welded joints. At present, the post-weld rolling treatments for strengthening age-hardening aluminum alloy joints mainly include the following two types:Du et al.  employed(2)Post-weld partial rolling: After welding with a filler wire, only the weld reinforcement is rolled , as shown in Song et al.  found thThe over-aging phenomenon of the strengthening phase occurs during the welding process of age-hardening aluminum alloy, which seriously reduces the mechanical properties of the welded joint. The softening range and degree of strengthening phase in the joint are closely related to the welding heat input. Compared with traditional TIG, MIG and other large-heat-input welding heat sources, LBW, FSW and CMT, with low heat input, have more significant advantages in the welding of age-hardening aluminum alloys, as they can greatly reduce the softening degree and range of welded joints and improve the performance of joints. In addition, welding researchers also found that the softening of the joint is related not only to the peak temperature range during welding, but also to the residence time at high temperatures. Therefore, the externally assisted cooling technique is adopted in the welding process to reduce the peak temperature range and increase the cooling rate, so as to control the influence of the welding thermal cycle on the strengthening phase. In most cases, the mechanical properties of the joint can be further improved. Although the low-heat-input welding method and externally assisted cooling process improve the joint softening of age-hardening aluminum alloys, the softening of the weld seam and heat-affected zone remain and often represent a weakness in the service process. Therefore, strengthening the post-weld joint softening zone is of great significance to improve the joint performance. In current studies, researchers mainly adopt post-weld heat treatment, post-weld surface modification treatment and post-weld rolling, through precipitation strengthening by the reprecipitation of the coherent strengthening phase, strain strengthening by introducing dislocation or both to improve the properties of the softening zones. However, currently, PWHT is still considered the best method to improve the properties of the soft zone, especially for thick-plate joints. However, due to the differences in the base metal chemical compositions, initial temper conditions and thickness, welding methods and process parameters, the optimal heat treatment process of the joint will also be different. PWHT for joints welded by dissimilar aluminum alloys is a challenge, and research is ongoing. This is due to the different base metals requiring different optimal treatment processes, particularly for the peak-aging processes of base metals, which differ significantly.(1)Further research on the softening mechanism of aluminum alloys should be carried out to establish the relationship between the welding thermal cycle and joint grain size, strengthening phase characteristics, dislocation density and texture, in order to transform the analysis of the effect of welding heat input on joint softening and performance from qualitative analysis to quantitative analysis.(2)It is necessary to establish the precise response mechanism of an over-aged microstructure and the temperature field of a welded joint, monitor the state of the welding heat source and the information about the plate temperature field in real time and dynamically and scientifically control the parameters of the welding heat source, in order to precisely control the shape and performance of joints during the welding process.(3)According to the softening characteristics of the joint, it is necessary to develop post-weld local strengthening treatments to achieve the cooperative strengthening and toughening of the properties of each zone of the welded joint.(4)It is necessary to establish the relationship between the softening characteristics of the joint and fatigue performance, corrosion performance and forming performance, to promote the research and development of age-hardening aluminum alloy tailor-welded blank manufacturing technology and to expand the scope of application of the materials.(5)It is necessary to research the evolution of the microstructure and properties in the softening zone of welded joints and the strengthening of joints over time, to provide theoretical and data support for evaluating the service performance of structural parts.Age-hardening aluminum alloy is a kind of non-ferrous metal that can meet the worldwide requirements of energy-saving, emission reduction and light weight. However, the precipitation strengthening effect is easily affected by the welding heat input. To improve the performance of joints welded by conventional fusion welding, which results in a large welding softening range and severe softening degree, researchers have adopted many effective welding processes and methods, such as low-heat-input welding methods, externally assisted cooling techniques and post-weld treatment techniques. Although studies have made great achievements, the joint performance remains lower than that of the base metal and there is room for improvement. In addition, PWHT for dissimilar age-hardening aluminum alloys is a challenge owing to the different base metals requiring different optimal treatment processes. Therefore, it is necessary to carry out further study as follows:


Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and are responsible for botulism, a fatal disorder of the nervous system mostly induced by food poisoning. Despite being one of the most potent families of poisonous substances, BoNTs are used for both aesthetic and therapeutic indications from cosmetic reduction of wrinkles to treatment of movement disorders. The increasing understanding of the biology of BoNTs and the availability of distinct toxin serotypes and subtypes offer the prospect of expanding the range of indications for these toxins. Engineering of BoNTs is considered to provide a new avenue for improving safety and clinical benefit from these neurotoxins. Robust, high-throughput, and cost-effective assays for BoNTs activity, yet highly relevant to the human physiology, have become indispensable for a successful translation of engineered BoNTs to the clinic. This review presents an emerging family of cell-based assays that take advantage of newly developed human pluripotent stem cells and neuronal function analyses technologies. Botulism is a potentially fatal disorder of the nervous system affecting both humans and animals, most often after ingestion of food contaminated with bacteria or spores. Botulism results in progressive flaccid paralysis of motor and autonomic nerves. It was first described in the 1820s by Kerner, who presented a study on several patients suffering from fatal poisoning after ingestion of contaminated sausages. Kerner described in these patients muscular paralysis of respiratory muscles, muscles of the upper and lower limbs, and vegetative disorders such as mydriasis, double vision, and gastrointestinal and bladder disorders . “KernerIn 1895, Ermengem isolated for the first time an anaerobic bacterium, called Clostridium botulinum, from a contaminated ham as well as the intestine and spleen of botulinic patients ,5. AfterDespite being considered as one of the most poisonous substance known, with a lethal dose estimated at 0.1–1 ng/Kg in intravenous and 1 µg/Kg orally in Humans, botulinum neurotoxins (BoNTs) have, over the years, successfully become a therapeutic agent for a wide spectrum of disorders especially neurological disorders when injected locally, as well as for aesthetic applications ,15,16,17The recent advances in stem cell biology have raised expectations for BoNTs research, especially in light of the recent developments in human pluripotent stem cells technologies. Human pluripotent stem cells (hPSCs) are characterized by two main cardinal properties: a capacity to give rise to all the cell type forming an organism, and the capacity to self-renew almost without any limitation. When combined together, both properties of hPSCs offer, in theory, unlimited access to highly relevant cell source for a range of applications in drug discovery. Two sources of hPSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have shown the most promise in the fields of regenerative and transplant medicine, disease modeling, high-throughput screens for drug discovery and development, and human developmental biology .In this review, we examine recent advances in cellular models for the study of BoNTs most particularly those based on hPSCs. We describe their suitability for high-throughput studies including drug screenings aimed at identifying and evaluating novel BoNT-based therapeutics. The design of high-content stem cell-based models has the potential to extend our understanding of BoNTs intoxication process and help harness therapeutic potential of possibly new classes of compounds based on BoNTs. As the development of BoNT-based compounds is often marred by the lack of physiologically relevant and predictive assays, the use of stem cell-based models offers great opportunity as a valuable and a sensitive system to study biological effects of BoNT in a time and cost-efficient manner. Stem cell models can, under many conditions, provide an alternative to the animal models generally used in the field .In the last decades, a growing number of studies have contributed to describe the mechanisms by which the BoNTs block neuromuscular transmission ,21, the 2+)-dependent manner. The cleavage of neuronal SNARE proteins by BoNT prevents membrane fusion and blocks the release of cholinergic neurotransmitters at the neuromuscular junction, ultimately leading to neuroparalysis.BoNT in its native form is protected by multi-protein complexes composed of proteins associated with non-toxic neurotoxins, the Non-Toxic Non-Hemagglutinin (NTNHA) proteins ,27. ThesNeutralizing antibodies can distinguish at least seven different serotypes of BoNT, from A to G, that are structurally similar but antigenically distinct. Several studies reported that monoclonal antibodies can neutralize only some but not all serotypes suggesting variability within each neurotoxin serotype. Such variabilities are now revealed and explained through sequencing of neurotoxin-encoding genes of bacteria infecting patient . A BoNT 2+ level triggers the membrane fusion activity of SNARE complexes, resulting in exocytosis of their content, the acetylcholine (ACh), into synaptic cleft. ACh then binds to its receptor on the surface of the muscle fiber triggering a new nerve impulse that spreads rapidly along the muscle fiber to make it contract [Physiologically, the neuromuscular transmission starts when a nerve impulse from peripheral or central nervous system reaches the nerve terminal of the neuromuscular junction (NMJ). At the synapse, the depolarization of the presynaptic membrane triggers the opening of voltage-gated calcium channels. This increase of intracellular free Cacontract ,66. Uponcontract ,69,70,71The mechanism of action of BoNTs first involves their binding to receptors in the presynaptic membrane of nerve terminals. The active site of binding localized in the C-terminal HC portion of BoNTs, recognizes two distinct types of receptors simultaneously. The first type corresponds to gangliosides receptors abundantly expressed on neuronal plasma membrane and known to interact with BoNT with low affinity ,73. The The direct consequence of the inhibition of ACh release is a reduction in muscle contractions and efferent signaling at the NMJ, ultimately causing neuroparalysis. This inhibition of synaptic transmission is temporary and fully reversible, because a synaptic remodeling and a recovery process takes place at the NMJ which restores synaptic contacts and rescues functional neurotransmitter release ,75,76. IBoNTs present significant opportunities to identify new ways to treat various clinical indications when used in low concentrations. Proper choice of dosage and site of administration are essential determinants of a positive beneficial effect to BoNTs treatment. Despite the great diversity of natural BoNTs, only serotypes A and B are commercially available so far. In addition to being approved by the US Food and Drug Administration (FDA) , BoNTs hThe natural diversity of BoNTs as well as the increasing knowledge of their activities provide the opportunity to address novel clinical applications. In this context, the development of new cell-based assays to study biological effects of these neurotoxins may help to reduce the time and cost of current models. Several are undergoing investigations and clinical trials test the efficiency of BoNTs for new therapeutic indications, i.e., epilepsy , tremor Currently, the standard assay to test the presence of BoNTs relies on the in vivo mouse bioassay (MBA) . This asDespite these advantages, several issues can be raised concerning the “3 R’s rule”: Reduce, Refine, Replace. First ethical considerations for the well-being of the animals have been raised due to the large number of mice needed for such assays ,92. The In order to overcome these limitations, efforts have been made to develop alternative solutions. For example, the nonlethal mouse Digit Abduction Score (DAS), recently adapted to rats, has been developed to detect and identify BoNTs serotype after intramuscular injection into lower limb skeletal muscle ,101. NonOther approaches to test BoNTs based on ex vivo models have been explored. They consist in a continuous measurement of the twitch force elicited by electric stimulation of an isolated explant of adult rodent muscles and nerves in a physiological bath. The mouse or rat phrenic nerve hemidiaphragm (PNHD) assay uses isolated hemidiaphragm muscle with the attached phrenic nerve from euthanized rodents ,102,103.Altogether, ex vivo assays prove particularly useful to decipher BoNTs mechanism of action and identify intracellular events and receptors involved in neurotoxicity. Although ex vivo assays can replace the MBA, they are still based on the use of animals, and they require the implementation of expensive technical platforms necessary for all the measurements.In the last decade, significant progress in the assessment of BoNTs’ potency has been made toward reducing animal use. Different cell-based in vitro assays (CBA) using specific neuronal cell lines have been implemented to detect multiple steps of BoNTs activity including membrane receptor binding, toxin uptake, translocation, and intracellular substrate cleavage ,105. TheOne of the main read-outs used in CBA is SNAP25 cleavage which can be quantified either by Western Blotting ,107, ELI® in 2011, XEOMIN® in 2015, DYSPORT® in 2018), which supports the relevance of such assays in this field.Furthermore, in the field of BoNTs research and development, for several years commercial batches of toxins have been validated with cell-based assays  these cells self-renew in culture without limitations and can thus be produced in large quantities, (ii) they are easy-to-use, and (iii) relatively inexpensive ,114,115.Primary neurons can be obtained from different embryonic neural tissues, including spinal cord , dorsal Since their discovery more than 20 years ago, hPSCs have ushered in a new era for the fields of stem cell biology and regenerative medicine, as well as disease modeling and drug discovery.The “physiological” and “natural” source of human pluripotent stem cells are the human embryonic stem cells (hESCs), which are derived from human blastocyst of in vitro fertilized embryo . While hThe main conditions for hPSC-based therapeutic development rely on the “holy grail” to efficiently and robustly differentiate hPSCs into all cell types of interest. Much effort has focused on the differentiation of hiPSCs into mature cell types and the last decades have witnessed the development of more and more robust and efficient protocols allowing the conversion of hiPSCs into a large panel of different cell types such as pancreatic beta cells, hematopoietic cells, cardiomyocytes, skeletal muscle cells and neural cells. Regarding the latter, relevant protocols have been developed raising the possibility to trigger neural induction  and nextThe derivation of hPSCs regardless their origin sparked enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of cell-based therapy. Several pioneering studies have validated the potential of disease-specific hESCs to reflect diseases intrinsic to the cellular level ,151,152.In order to exploit the therapeutic potential of BoNTs, appropriate in vitro models are needed to extend the knowledge about mechanism of action, to characterize and compare serotypes, and support translation to the clinic.There are currently few studies in the literature focusing on the use of hiPSCs for the study of BoNTs . AlthougThe use of iPSCs of human origin should increase species-specific relevance and offer high sensitivity together with the possibility to compare BoNT serotypes . Robust 2+ within and between connected cells, the electrical activity, the activity of part of the system, the connection between the cells composing the system, and the activation or inhibition of a part of the system.Many studies confirmed that iPSCs-derived neurons  express all the necessary receptors and substrates for BoNT intoxication, and are sensitive for detection of several BoNTs serotypes with different potencies, making iPSC-derivatives cells of choice for BoNT research ,162,163.The development of hiPSC-based systems and the progress made in recent years on increasingly robust hiPSCs differentiation protocols has resulted in the creation of more physiological and functional models for drug screening in miniaturized format to build attractive systems that are translational for preclinic and clinic research. Combined with the progress made in high-throughput technologies, it is possible to evaluate more complex and precise functional parameters that should open new perspectives in the field of BoNTs. In this section, we will evaluate these perspectives notably in terms of functional analyses as well as the challenges that still need to be overcome.Properties of ion channel function are routinely measured by patch clamp which is considered the gold standard for analysis of membrane electrical activity. Considerable effort to automate this analysis has been made for pharmacological testing of compounds to evaluate the potency of compounds-channel interactions. Progress has been achieved on the miniaturization and automation of the electrophysiological set up to increase the throughput and sensitivity of simultaneous recording of single cell, and assess cumulative or multiple compound additions ,176. A mIn the area of BoNT research, channel-specific toxins have multiple mechanisms of action, including interference of ion-channel opening. BoNT binding alters normal conformational changes required to open or close activated channels which can cause uncontrolled neuronal excitation, increased or reduced neurotransmitter release, and muscle spasm. BoNTs offer an example of toxin-induced structural change causing alterations in neuronal network electrophysiology by reducing or interrupting synaptic signals . SeveralTo respond to these challenges, one possible alternative may reside in the use of multi-well microelectrode arrays (MEA). Whereas patch clamp electrophysiology records the action potential electrical activity from the intracellular space of a single neuron. MEA is a noninvasive electrophysiology technique that records the field potential electrical activity from the extracellular space of a population of neurons.In the context of BoNT research, the combination of MEA systems and stem cells has recently been successfully applied to evaluate potency and efficiency of BoNT/A on neuronal cultures derived from mouse ESCs . In thisIn parallel to these electrophysiological approaches, the recent development of optogenetic techniques has generated considerable excitement in neuroscience research. Optogenetics, first developed in 2005, is a non-invasive genetic method that involves the use of light to control the activity of cells with both high temporal and high spatial precision . As optoRecently, BoNTs have been the subject of optogenetic engineering. Liu and colleagues described a blue light activatable BoNT engineered to disrupt excitatory neurotransmission resulting in persistent synaptic inhibition triggered by light . Using tAlthough hiPSC-derived systems are relevant tools with considerable advantages for studying BoNTs, including for high-throughput analyses, several concerns can still be raised on the relevance of these systems. First, the developmental stage of hiPSC-derived neurons remains unclear. In general, in vitro disease modeling techniques use short-term cultures that fall short of producing really mature hiPSC-derived neurons. To overcome this limitation, approaches such as prolonged neural differentiation , long-teAnother important concern resides in the fact that BoNT intoxication in humans arises in the context of complex multicellular tissues and organ systems and is dependent on multiple interactions occurring between cells, extracellular matrices, and pathogens. Consequently, more complex iPSCs-based differentiation systems  are currently being developed to faithfully recapitulate human tissue-level and organ-level dysfunctions ,197,198.Significant advance in the generation of 2D culture systems and recently in 3D culture systems open new perspectives for BoNTs research . While 2Faustino Martins and colleagues established a 3D human neuromuscular organoid system that self-organized and formed functional NMJ . Muscle The 3D models provide important information in understanding several mechanisms underlying neuronal network function and homeostasis, but they do not fully replicate the exact human physiological functional units formed by the connection of different excitable cell types, resulting from separated cellular microenvironments. To address this need, compartmentalized microfluidic technology represents an alternative to 3D system to recreate NMJ physiology of human in vitro. Bellman and coworkers, as well as Osaki and colleagues, developed a microfluidic systems to model NMJ by creating a specific cell microenvironment for hPSC-derived motor neurons and hPSC-derived muscle cells ,210. SucDespite the fact that synapses including the NMJ are complex multi-cellular 3D structures, the majority of drug screening and safety or efficacy measurement are still carried out using mono-culture of cells grown a 2D monolayer ,212. NotRecent studies have established the versatility of hPSCs to generate clinically and biologically relevant multicellular models. For BoNT detection and mechanistic studies, various cell models currently exist, but none examine BoNT function with human-specific relevance while exhibiting high sensitivity.To study BoNTs, preferential models have been developed over the years: in vivo, BoNT-induced muscle relaxation is assessed in mice and rats (MBA); BoNT potency was evaluated ex vivo on mice hemidiaphragms, measuring muscle contractility; and BoNT potency was preferentially assessed in vitro in rodent primary cells (CBA). All these models have several limitations such as the requirement for significant technical expertise and highly skilled personnel to implement the assays, reproducibility and stability, cross species differences limiting translation of data from animals; they also raise important ethical concerns regarding the large number of rodents require by these assays. In this context, more physiologically and predictively relevant models are needed. Human iPSCs technology offers the opportunity to study BoNTs in relevant humanized context making cell-based assays crucial for the advancement of BoNT research and drug discovery. The continuous development of robust protocols for the differentiation of hiPSCs into various neuronal sub-populations opens the door to comparative studies of different BoNTs serotypes in different neuronal sub-populations. Several studies have shown that hiPSC-derived neurons were highly BoNT-sensitive models with different sensitivities depending on the BoNTs serotypes. Moreover, the generation of a variety of neurons of different phenotypes  allows the development of more complex, translational, and predictive cell-based systems to help in the development of a panel of BoNT-based future therapeutics in the areas of movement disorders, neurological diseases, neurodegenerative diseases, pain, and other disorders.The complexity, in terms of the number and interconnectivity of cell types, maturity, and differentiated state of most cells of the human nervous system is difficult to replicate in cell-based models. One important aspect of cell-based assay is the read-outs often provide information on cell viability, morphology, and neuron-specific functional activity in addition to the primary output. Progress in high-throughput assays can provide information on multiple functional parameters such as electrical activity, network synchrony, calcium transient. In this sense, human neuronal cells derived from iPSCs are attractive models because they exhibit the function, connectivity, and behavior of mature neurons in a synaptic network.To identify network activity in a high-content manner, multiple laboratories have employed electrophysiology, MEA, and more recently optogenetics platforms to evaluate in a high-content context the activity of a neuronal network by using relevant read-outs such as calcium signaling. Several studies showed the efficacy of these techniques to record the activity of synaptic networks which is an emerging target for the development of future therapeutics. Taking advantage of hiPSCs-derived systems, sophistication of models , and high-throughput functional assays, the study of the effect of different BoNTs serotypes on synapses such as NMJ, but also on other neuronal synapses, should open the way to new BoNT-based therapeutics.


Plants are the prime source of vital secondary metabolites (SMs) which are medicinally important for drug development, and these secondary metabolites are often used by plants in the various important tasks like defense against herbivory, interspecies defenses and against different types of stresses. For humans, these secondary metabolites are important as medicines, pigments, flavorings and drugs. Because most of the pharmaceutical industries are highly dependent on medicinal plants and their extraction, these medicinal plants are getting endangered.Plant cell culture technologies are introduced as a viable mechanism for producing and studying SMs of plants. Various types of in vitro strategies  have been considerably used for the improvement of the production of SMs of plants. For the enhancement of SM production, suspension culture and elicitation are mainly used, but hairy root culture and other organ cultures are proved to satisfy the demand of secondary metabolites. Now, it is easy to control and manipulate the pathways that produce the plant secondary metabolites.Techniques like plant cell, tissue and organ cultures provide a valuable method for the production of medicinally significant SMs. In recent years, most of the in vitro strategies are used due to knowledge and regulation of SM pathway in commercially valuable plants. In future, these things will provide a valuable method to sustain the feasibility of medicinal plants as the renewable sources of medicinally important compounds, and these methods will provide successful production of desired, important, valuable and also unknown compounds. Plants are the valuable source of medicines that play a vital role in the mitigation of overall global health issues , but some plant species fail to produce significant amounts of secondary metabolites in suspension culture  . This is why plant cell, tissue and organ cultures have been proved an appropriate alternative for the SMs production (Rao and Ravishankar a) Ellis .Plant cell and tissue cultures are another possible industrial method for the secondary metabolites production , glycoproteins, G-protein and intracellular proteins. Exogenous elicitors are those compounds which are formed exterior to the cell, e.g., polysaccharides, polyamines and fatty acids, whereas ‘endogenous elicitors’ are those types of compounds which are formed inside the cell, e.g., galacturonide or hepta-β-glucosides, etc.  and abiotic factors  for which it transformed its genes in plant’s genome. This transformation causes the development of hairy root at the infection site of host plant . Once bacterium attached or colonized to the wounded plant cells, it leads to the insertion of T-DNA fragment of Ti-plasmid (in case of A. tumefaciens) or Ri-plasmid (in case of A. rhizogenes) to the host plant cells and integrated into the plant genome. Many vir genes located in 40-kb region of Ti-plasmid or Ri-plasmid called the virulence (vir) region play significant characters in the Agrobacterium-mediated transformation process. The roles of Vir have been considered in a number of outstanding review articles  contain two autonomous T-DNA represented as TL-DNA (left-handed T-DNA) and TR (right-handed T-DNA). Both the TR-DNA and TL-DNA have high resemblance to the T-DNA of the Ti-plasmid of A. tumefaciens and Ri-plasmid of A. rhizogenes strains, respectively  induce roots to produce agropine, mannopine and corresponding acids.Mannopine-type strains  elicit roots containing only mannopine, mannopinic acid and agropinic acid.However, Zhou et al.  classifiIn plant tissue culture, hairy root culture exhibits the greatest advantage for biosynthetic capacity of SMs production related to their mother plants   techniques and optimizing their large-scale production using bioreactors. PTC techniques being independent of climatic and geographical conditions will provide an incessant, sustainable, economical and viable production of secondary metabolites  for the particular plant species. Oxidative stress plays an important role in the production of secondary metabolites in plants. Phenolic compounds are considered to be secondary metabolites that are synthesized in plants through the phenylpropanoid pathway and function as a defense mechanism that reacts to various biotic and abiotic stress conditions. The exposure of plants to unfavorable conditions leads to the generation of reactive oxygen species (ROS). The well-organized way to enhance the secondary metabolite production in callus suspension cultures was deeply analyzed in Agrobacterium rhizogenes. In some cases, hairy root cultures are considered most reliable than cell cultures for the commercial production of SMs. Shoot cultures and hairy root culture are useful because they provide a constant and reliable basis for the SMs production. The other advantage of these techniques is that they are independent of various geographical, seasonal and environmental conditions. Biotransformation helps in discovering the new compounds for pharmacological activity and for other compounds modifying their chemical structures allowing them to show pharmacological activities. In addition, biotransformation also provides synthesis of new compound in suspension culture of medicinally important plants. The use of genetic engineering, regulation of biosynthetic pathways of desired plant metabolites offers the production of commercially valuable secondary metabolites. Many molecular biology techniques, which are used in tissue cultures, induced the SM production by effecting the expression and regulation of biosynthetic pathways. In recent years, most of the in vitro strategies are used due to knowledge and regulation of SM pathway in commercially valuable plants. In future, these techniques will provide a valuable method to sustained feasibility of medicinal plant as renewable source of medicinally important compounds and these methods will provide successful production of desired, important, valuable and also unknown compounds.Medicinal plants are important source of SMs which are directly or indirectly used by various pharmaceutical industries due to which the supply of these SMs is limited. The limited availability of biologically active, commercially valuable and medicinally important plant SMs can be overcome by using metabolic engineering and biotechnological processes. Advances in these techniques, particularly plant cell, tissue and organ cultures, provide valuable method for the production of medicinally important SMs. In cell cultures, suspension culture and elicitation are important strategy to enhance the SMs production. In various plants, these methods proved to be valuable tool for the production of SMs. Hairy root culture is an important method for enhancement of SMs production by using


Copy number variations (CNVs) are the predominant class of structural genomic variations involved in the processes of evolutionary adaptation, genomic disorders, and disease progression. Compared with single-nucleotide variants, there have been challenges associated with the detection of CNVs owing to their diverse sizes. However, the field has seen significant progress in the past 20–30 years. This has been made possible due to the rapid development of molecular diagnostic methods which ensure a more detailed view of the genome structure, further complemented by recent advances in computational methods. Here, we review the major approaches that have been used to routinely detect CNVs, ranging from cytogenetics to the latest sequencing technologies, and then cover their specific features. Ever since Joe Hin Tjio and Albert Levan first identified the exact number of chromosomes in human cells in 1956, there have been massive advances in the understanding of the human genome and its structure ,3. It waHence, the aim of this review is to highlight the major contributions made in the field to detect CNVs to date and describe their main principles and challenges. We will also describe the most widely used computational approaches and discuss burning issues in this research field.The first approaches to CNV detection were based on the analysis of metaphase plates in cells. During metaphase in cell division, condensed chromosomes align along the cell equator, facilitating convenient viewing of chromosome structure through light microscopy. Until the early 1970s, the only method for chromosome staining was Giemsa staining. In such an approach, all chromosomes are stained evenly length-wise, allowing for changes in karyotype concerning chromosome number, shape, and size to be visualized. Giemsa staining was used to detect many chromosome aneuploidies, such as Down’s syndrome , KlinefeAfter the advent of Giemsa staining, new methods of differential staining that allowed for visualization of certain chromosomal structures, e.g., C-banding for centromere or T-banding for telomere regions staining, as well as producing chromosome-specific sequences of dark and light bands along the length  were developed. The most common was the G-banding technique, a method that implies preliminary treatment of chromosomes with trypsin for DNA denaturing and further staining of renatured chromosomes with Giemsa dye . Pairs oMore detailed analyses of the genome became possible upon the development of DNA hybridization techniques. At first, tritium-labeled RNA probes were used to search for a specific DNA sequence and the hybrids were detected by autoradiography . Later, For whole-genome analysis, comparative genome hybridization was developed in 1992 . In contThe application of microarrays instead of metaphase chromosomes has provided a solution for large-scale genome studies. Hybridization occurs on multiple DNA probes attached to a solid surface, and physical position on a chip and specific nucleotide sequences of the probes are pre-determined, which allows visualization of relative fluorescence intensities of test and control samples along the genome. For example, in 1997, the matrix-based comparative genomic hybridization method was introduced , and sooFurther improvement of the method occurred upon the deciphering of the whole-genome sequence. This development led to the application of oligonucleotide probes (8–25 bp) that had been previously used for gene expression studies ,34. OligAnother type of microarray used to search for regions of chromosome imbalance has been developed for genotyping and genome-wide association studies. Here, only fluorescently labeled DNA of the sample is applied to the chip, and copy number analysis is performed based on the rate of target DNA hybridization to allele-specific probes b. Such aNot all chip platforms are equally fit for CNV detection. Although for any type of microarray  there is a dependence of the number of identified variations on the total number of probes, the design plays an equally important role . HaraksiThe aim of any algorithm is the analysis of signal intensity along the genome (logR) and elucidating any significant deviations. For this purpose, both simple thresholds, e.g., change of logR by 0.2–0.3 ,44,45 orA large class of algorithms utilize hidden Markov models (HMM), allowing for the description of a system with unknown variables based on the observed ones. Changes in the number of copies   or absolToday, multiple ways for microarray data to be processed have been proposed . They diMethods of CNV detection gained momentum with the rise of high-throughput sequencing  technology. Based on the analysis of millions of short readings, this was quickly deemed a revolutionary technique due to its high productivity, reproducibility, and accuracy. Data generated from this technique can also be used repeatedly in various types of studies, and this research domain has expanded considerably concerning some genomic variations that were previously difficult to detect. Firstly, the microarray technique had limitations in accessing balanced rearrangements. Secondly, as a consequence of multiple sequencing of random fragments, variation size is not strictly limited as is the case in CMA, where variation less than the distance between two probes cannot be resolved.Detection algorithms are readily being developed in several directions since NGS data can be described by various signatures . One of The split-read (SR) approach also originated from the incoherence property; however, in this case, read is not mapped onto the genome at all or is only partially mapped. The following repeated alignment of the read parts can indicate possible coordinates of the start and end of the variation . The SR In contrast to previous methods indicating only the presence of a variation, the next approach was intended to evaluate the number of copies. The read-depth (RD) approach is based on the assumption that region coverage correlates with the number of its copies. Not limited by either read length or insert size, the approach is suitable for the identification of preferably large variations. The standard detection procedure consists of four stages: mapping of reads and calculation of coverage; normalization; segmentation; and evaluation of the copy number. In the first stage, a genome, or a sliding window, is usually used. The window size can be both chosen voluntarily  or seleThe RD approach is applicable to the data of whole-exome or targeted sequencing . Althoughttps://github.com/broadinstitute/gatk/blob/master/docs/CNV/germline-cnv-caller-model.pdf, accessed on 9 February 2022). A somatic mode is also available.Recently, the team behind the GATK , which is known as the most popular tool for analyzing short genomic variations, has also proposed a pipeline to call rare and common germline copy number variants. It uses negative-binomial factor analysis and HMM, and requires at least one-hundred samples to build a model . The approach does not require high coverage and is potentially fit for the identification of all types of structural variations, especially new ones. The assembly is performed using graph models (overlapping graphs built by the overlap layout consensus (OLC) method and de Bruijn graph  are mostDespite progress, each of the four described approaches alone is not able to identify the whole range of variations; therefore, the next step was the development of combined methods . The mosOften, CNV detection is narrowed down to classification problems solved by methods of machine learning. Along with various signatures, a number of additional factors, for example, mapping quality (MAPQ) or nucleotide content, are considered . In addiIntegration of the data can proceed not only at the level of the signatures, but also at the level of the variants predicted by multiple individual algorithms. Today, the so-called ensemble-based approach is not standardized in any way. Various methods are used for combining and evaluating the variants, including coordinate overlapping, distance between the breakpoints, signature prioritization, agreement of the algorithms, number of confirmations of the event, confidence intervals of the breakpoints, FDR cutoff, and metaheuristics ,112,113.On the contrary, long reads (above 5 kb) produced by third-generation sequencing machines can be used to solve the problem, despite their lack of accuracy. The Pacbio platform performs real-time sequencing of a single molecule through the synthesis of a new strand using a polymerase bound to the well bottom and registration of fluorescence of each newly added nucleotide. In turn, the Oxford nanopore technology is based on the evaluation of change in an electrical current induced by a single-stranded DNA molecule passing through a nanopore. In either case, to detect variations, signatures inside the reads completely covering variations and signatures indicating the presence of a variation based on discrepancies between the reads  are analyzed ,115 MoreAs an alternative to long reads, the sequencing of bound molecules can be used. For example, using additional barcodes to indicate association to a single DNA molecule, synthetic long reads can be efficient in the detection of large variations. The identification of variants proceeds based on the evaluation of the density of molecule coverage by paired reads, the excessive increase or decrease of which implies a CNV event in a certain region ,118. In Saccharomyces cerevisiae chromosomes has undergone some modifications [In addition to the sequence-based methods mentioned above, optical mapping that aims to determine the physical location of specific sequence motifs or enzymes has great potential for CNV calling. The method first demonstrated in 1993 on the example of construction restriction maps of ications . Today’sications , haplotyications , and seaications . A CNV eications .Despite considerable progress, the identification of copy number variations remains a difficult task. Each of the proposed approaches, from cytogenetics to emerging sequencing technologies, capable of the analysis of the so-called dark genome regions has its own limitations. In an attempt to neutralize the latter, over one-hundred methods of analysis have been developed. Particular attention is paid to the development of algorithms aimed at exome and targeted sequencing as optimal tools for applied methods of genome analysis in regard to information load and cost , as wellEvaluations of the efficiency of existing CNV detection methods and understanding their advantages and limitations are complicated by the lack of a comprehensive validation set. The only available approach for the description of many genomic variations today is the integration of the results of several platforms, as proposed by Zook et al., for simple deletions and insertions . Expansi


Nowadays, severe symptomatic tricuspid regurgitation (TR) affects millions of persons worldwide. However, the benefit of surgical correction of isolated secondary TR remains controversial because of the increased risk of periprocedural mortality and morbidity. In recent years, novel transcatheter tricuspid valve interventions (TTVI) were developed to treat TR, so that TTVI is currently considered in symptomatic, inoperable, anatomically eligible patients. TTVI can be divided into these five domains: edge-to-edge leaflet repair, tricuspid annuloplasty, caval implants, spacer, and total valve replacement. Each transcatheter intervention needs specific imaging protocols for assessing the anatomical feasibility and consequentially predicting the procedural success. This review summarizes the available multimodality imaging tools for screening patients with TR, and identifies anatomical characteristics to choose the best option for the patient. Interest in the tricuspid valve (TV) has increased over the past two decades, because of the recognition of the progressive nature and clinical impact of secondary tricuspid regurgitation (TR) on outcomes , 2. FurtIn recent years, novel transcatheter tricuspid valve interventions (TTVI) were developed for treating TR, so that the current european society of cardiology (ESC) and the european association for cardio-thoracic surgery (EACTS) guidelines for the management of valvular heart disease consider TTVI in symptomatic and inoperable patients who met anatomical criteria for eligibility . NumerouThis article reviews the TV anatomy and the available imaging tools for a comprehensive assessment of the valve. Finally, it identifies favorable and unfavorable anatomical conditions that can guide the operator to choose the best option for the patient.2 and the most apically located valve. Its functional anatomy consists of four elements named annulus, leaflets, papillary muscles, and chords  reconstruction of the tricuspid annulus showed a non-planar saddle-shaped pattern with two high points, corresponding to the AS and post-erolateral segments, and two low points corresponding to the anterolateral and PS segments . The annIn patients with functional TR, the tricuspid annulus is flatter, which reduces the saddle shape. Compared with normal subjects, patients with functional TR have a more circular annulus, and greater enlargement in the AP than in the mediolateral distance, because the greater dilation occurs along the free-wall side of the annulus. In presence of TR, the percent reduction of the tricuspid annulus size during the systole is also significantly decreased. Therefore, the minimum systolic annular dimension is almost twice larger than in normal subjects .Characterization of the anatomy of the RV and TV is the first step in the evaluation of patients with functional TR, candidates for TTVI. While historically the anatomical knowledge of TV was derived from cadaveric specimens or open, today non-invasive imaging techniques represent helpful instruments to understand the anatomy of cardiac structures. Currently, echocardiography, CT, and cardiac magnetic resonance (CMR) are available instruments to study the TV anatomy, guide in the decision-making process, and support the development of novel transcatheter therapies. In addition, the current ESC/EACTS guidelines for the management of valvular heart disease recommend transesophageal echocardiography (TEE) and cardiac CT for detailed anatomical evaluation owing to higher spatial resolution .Echocardiography is undoubtedly the first step in assessing the etiology and severity of TR and the size and function of the RVs. Moreover, this is the only imaging technique available for intraprocedural TTVI guiding. But the anterior location can pose challenges for the transesophageal approach. CMR is the gold standard for assessing right ventricular size and function and can help in quantifying TR when echocardiographic doubt exists. However, the reduced availability and contraindications limit the routine application of CMR in this setting of patients. On the other hand, CT provides complete morphologic imaging of the heart structures, thanks to a high spatial resolution with excellent capacity to define the endocardial border. However, disadvantages are the need for radiation and iodinated contrast injection.Transthoracic echocardiography (TTE) is the first-line approach for studying the anatomy and function of TV and right-side chambers . Due to Transesophageal echocardiography is the second-line imaging modality for the assessment of TV anatomy in patients with TR, even if the anterior position of the valve can limit the examination. Nevertheless, TEE allows obtaining imaging from several depths  and multiplane angles. The four-chamber view (0–0°) and the alternative four-chamber view (160–180°) permit the visualization of the septal and typically the anterior leaflet; the posterior leaflet can also be visualized by changing probe angulation. Simultaneous biplane imaging may clarify which leaflet is imaged because the anterior leaflet is usually adjacent to the aorta, whereas the coronary sinus is the marker for the posterior leaflet. Other useful information for a comprehensive TV assessment may be achieved by a mid/deep esophageal RV inflow/outflow view in multiplane (60–100°), which crosses the coaptation between the septal and the posterior leaflet  and the septal and anterior leaflet (near the aorta)  Figure .Then, advancing the TEE probe into the stomach results in transgastric views, which enables to obtain optimal view settings to :Identify all tricuspid leaflets and commissures. The transgastric short-axis view is the only 2D view that can simultaneously visualize all three TV leaflets;Visualize zones of coaptation and all coaptation gaps;Analyze the distribution of the TV regurgitation;Align the view parallel to the annulus of the TV and focus on the leaflets tips at closure, using the biplane modality.Three-dimensional echocardiography has greatly improved the accuracy of TV visualization and identification of all anatomical components, eliminating the need for mental reconstruction starting from multiple 2D planes . However2 as the most sensitive cutoff value for severe TR   (r = 0.97)  >0.75 cmy 82.1%) , whereasity 84%) . This vaity 84%) –20. More = 0.97) .2 RA and ventricular dimensionsanatomic relationships with surrounding structuresthe device landing zoneprediction of optimal fluoroscopic projections for device implantationvascular access route.A short-axis plane can be reconstructed on the annular level using multiplanar reconstruction starting from RV two- and four-chamber views. Cross-sectional area, perimeter, septolateral, and AP diameters can be obtained with excellent intra- and interobserver reproducibility . The croOnce the annulus has been manually or automated traced, commissures can be located, taking into account their unique anatomical findings. The fan-shaped chords can help in commissural identifications. Precisely, the AS commissure is placed anteriorly, just below the first right coronary tract and the anterior aortic valve cusp. At this site, it is possible to observe a short fan-shaped chorda, arising directly from the septal band of the crista supraventricularis or from a small papillary muscle on that band . The PS In the two- and four-chamber views, it is possible to obtain additional planes parallel to the TV for optimizing the visualization of the tricuspid leaflets. The grade of leaflet tethering, including tenting height, angle, and area can be obtained with a good to excellent intra- and interobserver variability . The degAdditionally, helpful information obtained by CT analysis is the AROA measurement, which may be used as a potential flow-independent anatomic parameter of TR severity, supplementing and enriching the traditional echocardiographic parameters , 36. ForMeasurements of the right chambers obtained with CT imaging may also be valuable in assessing the anatomic feasibility of a transcatheter therapy and planning the procedure.In a complete transfemoral approach, the RA must allocate the delivery catheter and have adequate space to permit safely catheter's movements. In case of transcatheter TV replacement, RV volume, as well as the distance between the annular plane and the RV apex and location of the papillary muscles are of interest with regard to the protrusion of the prosthesis into the RV. For quantification of RV volumes and function, semiautomated software can be used.Furthermore, assessment of the RCA course and its distance to the annulus, simulation of virtual device implantation, and prediction of an optimal projection angle are part of a comprehensive CT-based pre-procedural assessment  34)..34).Finally, the measurements of the inferior vena cava (IVC) along the vessels and at the transition level with the RA, as well as the anatomical relationship of the IVC and the annulus must be examined to plan the procedure. In the case of heterotopic caval valve implantation, additional measurements are obtained in regards to the first hepatic vein. Dedicated semiautomated software can be helpful in IVC analysis.Cardiac magnetic resonance is particularly well-suited for assessing cardiac morphology and function. It offers an excellent spatial resolution with the advantage of not using ionizing radiation and iodinate contrast agents. On the other hand, limitations in obtaining high-quality imaging and clear interpretation of reconstructions can occur in the presence of cardiac arrhythmias and intracardiac leads, which create significant artifacts.The RV size and function can be fully assessed using dedicated RV views from multiple planes. Moreover, CMR allows an accurate estimation of TR grade, in terms of regurgitant volume and regurgitant fraction, using direct (by right and left ventricular stroke volume) and indirect methods (by phase-contrast imaging). However, CMR quantification of TR severity is less established than for other regurgitant valves, and there are no specific cutoff values for severe TR , 37. AlsFurther studies are needed to evaluate the utility of CMR parameters, in defining the timing and predicting the response to TTVI.The TTVI can be divided into these five domains: edge-to-edge repair, tricuspid annuloplasty, caval implants, spacer, and total valve replacement . HoweverThe choice of the most suitable transcatheter therapeutic option in a single patient remains challenging , 39. ThiConsequently, a multimodality cardiac imaging focusing on the anatomy and function of TV apparatus, right-side chambers, and adjacent structures is mandatory for diagnosis and prognosis and the planning of transcatheter therapy.Indeed, specific imaging techniques for each transcatheter procedure are required to assess anatomic feasibility and select the correct procedure for an individual patient .Edge-to-edge repair devices act on tricuspid leaflets with the aim to improve the coaptation in the regurgitant valve. Currently available devices include the Triclip system , which grasps and coaptates leaflets , and theDuring the pre-procedural examination for edge-to-edge repair, a complete assessment of the TV anatomy is mandatory, including where the largest coaptation gap is located and which leaflets are more tethered. The echocardiographic 3D en-face view of the TV, the transgastric short axis, and the mid/deep esophageal RV inflow/outflow with simultaneous biplane view permit accurate assessment of the leaflets and commissures, as well as the largest coaptation gap. Preoperative CT imaging is not routinely performed for screening patients with TR who are candidates for edge-to-edge valve repair .Data from published experiences demonstrate that the best results occur by attaching the anterior and/or posterior leaflet to the septal leaflet, which can also reduce annular dimensions without distorting the valve . This zoThe favorable and unfavorable anatomical conditions for the edge-to-edge repair are listed in Annuloplasty devices aim to reduce the annular size in functional TR using a transfemoral ring- or suture-based approach. Precisely, the Cardioband (Edwards Lifesciences) consists of a flexible ring with multiple anchors attached to the annulus . The TriDetailed measurements of the tricuspid annulus are obtained by CT images. But the major concern related to annuloplasty devices is the risk of RCA injury as it runs closely and parallel to the tricuspid annulus. Therefore, it is imperative to routinely assess the course of the RCA and the distance between the vessel and the annulus by CT reconstruction. In addition, valve tethering should be assessed with both CT and echocardiography.The favorable and unfavorable anatomical conditions for the annuloplasty are listed in Heterotopic implantation of valves in the caval veins aims to reduce blood backflow and prevent systemic venous congestion. The technique counts balloon-expandable transcatheter aortic valve prostheses  implanted in the inferior (and superior) caval veins ; the TriThe most important parameters to consider in cardiac imaging for heterotopic implants include:Systolic backflow reversal in the inferior caval vein on echocardiography.Accurate measurement of the diameters of caval veins using CT. The IVC is measured at the level of the cavoatrial junction and at the level of the first hepatic vein. In addition, the distance between the cavoatrial junction and the most superior hepatic vein is measured to avoid obstruction of this vein during valve implantation. The superior vena cava is measured at the cavoatrial junction. In case of large RA, the cavoatrial junction may have a funnel shape, increasing the valve migration risk.Measurement of the RA is required for the Tricento device.Assess whether RV function is preserved because RA pressure increases and increased RV and atrial volume overload persist after the procedure, leading to further adverse remodeling and RV failure and preventing symptomatic benefit.The favorable and unfavorable anatomical conditions for the caval implant are listed in The Forma spacer device (Edwards Lifesciences) aims to minimize the leaflet coaptation gap caused by tricuspid annular dilatation and leaflet tethering, through a foam-filled polymer balloon (spacer) deployed at the level of the TV and an anchoring system placed in the RV septal free wall groove , 52.The pre-procedural imaging checklist includes:Measurement of the largest vena contracta or coaptation gap by echocardiographyMeasurement of the TV annulus, preferably from CTMeasurement of RV dimensions and distance between the annulus plane and RV septal free wall groove, using CT or CMRCT imaging of the proposed anchor site for possible obstruction to the passage of the rail and anchor systemDimensions of the left subclavian and axillary veins, which will be used to introduce the system. The CT is recommended to assess these measurements.The favorable and unfavorable anatomical conditions for the space technique are listed in Transcatheter TV replacement is a promising therapeutic tool that is still in its infancy. However, the procedure presents significant technical challenges due to the anatomical peculiarities of the TV annulus, which is enlarged, dynamic during the cardiac cycle, and without calcifications in the landing zone for the prosthesis. This can result in incomplete apposition of the prosthesis in the native annulus with consequent possible significant paravalvular regurgitation. Moreover, the anatomical relationship with surrounding cardiac structures  poses the risk of potential procedural complications. The NAVIGATE  is a self-expanding bioprosthesis for orthotopic replacement implanted through a transjugular or transatrial 42-Fr approach and consists of three xenogeneic pericardial leaflets, seated in a tapered nitinol stent with atrial winglets and ventricular graspers to anchor the tricuspid annulus and leaflets without protruding into the adjacent chambers. However, the short series published so far have shown the feasibility and safety of this procedure , 54. RecSpecific CT measurement protocols are needed for pre-procedural planning, including a comprehensive assessment of valvular and subvalvular anatomy, prosthesis sizing, vascular access route, and prediction of fluoroscopic angles. This study can be additionally supported by virtual prosthesis placement to predict implantation depth, prosthesis selection, and anchoring possibilities.Specifically, the pre-procedural imaging checklist should include :dimensions and morphology of the TV annulusRA and ventricular dimensionsdistance between the RV apex and the annular plane and location of the moderator band and prominent trabeculae with regard to the protrusion of the prosthesis into the RV during implantationanatomic relationships with surrounding structuresanalysis of vascular access route and relationship between caval veins and TV plane.On the other hand, in case of transcatheter TV-in-valve and valve-in-ring procedures, the measurements should be routinely assessed by a dedicated CT analysis rather than the nominal size documented in the surgical report.The favorable and unfavorable anatomical conditions for the orthotopic valve replacement are listed in With the ongoing rise in transcatheter TV interventions, procedural success extremely depends on precise and comprehensive pre- and periprocedural imaging. Multimodality imaging, including 2D and 3D echocardiography, as well as CT, is crucial in the complete anatomic evaluation of the right heart and TV, which are the target for current TTVI. On the other hand, the rapid growth and promising success of these procedures have been accompanied by advances in imaging techniques that have helped to accurately visualize the TV and its anatomic relationships, allowing appropriate selection of patients for the various techniques of transcatheter tricuspid repair.VC contributed with design and drafting of the manuscript. MC, MB, and SM contributed with revision of the content. GU contributed with conception, revision, and final approvement of the manuscript. FG contributed with conception of the paper. All authors agree to be accountable for the content of the work.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The concept of schizophrenia remains a matter of enduring debate, although often limited to the psycho-behavioral surface of its descriptive criteria. This Research Topic integrates such debate providing a set of different state of the art perspectives on the challenges surrounding the schizophrenia concept.In contemporary research, the notion of a broad psychotic spectrum, within which schizophrenia loses its nosological boundaries and dissolves, has gained once again a certain momentum. Proponents of this view argue that this concept is better supported by genetic, neurobiological, and neurodevelopmental data than the traditional categorically defined diagnoses. Others, however, defend the concept of schizophrenia or schizophrenia spectrum and continue the search for its essence that will allow demarcation of schizophrenia from other forms of psychosis. From yet other points of view, the Research Domain of Criteria (RDoC) and the Hierarchical Taxonomy of Psychopathology (HiTOP) address the general validity crisis of current nosography and explore new possible nosological horizons. This Research Topic aimed at an overview of possible nosological scenarios for schizophrenia and its spectrum disorders.Cuthbert and Morris, in their perspective article, argue that genomic data provide increasing support for the concept of systematic, trans-diagnostic components of neurodevelopmental and genomic spectra. In this view, the neurodevelopmental gradient is not simply a matter of cognitive performance, but a result of multiple functional domains, whose combinations comprise potentially significant clinical phenotypes with schizophrenia representing one segment of multiple broader spectra.Consistent with the RDoC approach, Cowan and Mittal, in their brief research report article, present a transdiagnostic dimensional analysis of psychiatric comorbidity in a Clinical High Risk (CHR) sample. They found that, although the CHR group presented more positive symptoms compared to healthy controls, the negative symptom factor was much more strongly linked than the other factors to impaired cognition, impaired social and role functioning, and risk of transition to psychosis This finding suggests that negative symptoms may be more specific of the progression toward psychosis in the broader spectrum of subthreshold positive psychopathology.From the perspective of HiTOP, Pontillo et al. found that in children and adolescents with early and very early onset schizophrenia, the presence or absence of neurodevelopmental disturbances or difficulties differentiated the characteristics of psychotic onset. In fact, in the presence of neurodevelopmental dysfunctions, the onset occurs earlier and was associated with more severe functional impairment, positive and disorganized symptoms. By contrast, in children and adolescents without neurodevelopmental disorders or difficulties, the psychotic onset was later and associated with negative symptoms.In their original research article, Collectively, these papers explore the relationship between neurodevelopmental disorders and schizophrenia through analysis of psychopathological trajectories and clinical pathways of childhood neuropsychiatric disorders.Gao et al. In their original research article, patients with schizophrenia showed an association between cognitive dysfunction and increased regional homogeneity values (ReHo), an index of neural activity, in prefrontal regions including the right rectus gyrus, inferior frontal gyrus/insula, the lower right and left insula, interestingly, all in the limbic area. Furthermore, ReHo values in the right inferior frontal gyrus/insula were correlated with negative symptoms and verbal learning tasks. The combined increases of ReHo values in the left inferior frontal gyrus/insula with the right gyrus rectus may be an underlying biomarker differentiating patients with schizophrenia from healthy controls.Exploring new potential phenotypes and endophenotypes, was another aim of the Research Topic, and it was addressed by Nordgaard et al. is the finding of a close relationship between formal thought disorders and self-disorders—a finding that further reinforces the notion of self-disorders as a unifying, psychopathological core beneath the apparently heterogeneous symptoms of schizophrenia spectrum disorders.Bleuler, who coined the schizophrenia concept, considered formal thought disorders (“disturbances of association”), and ego-disorders as fundamental symptoms of schizophrenia. In contemporary psychopathological research, ego-disorders have been re-conceptualized and systematically assessed under the notion of self-disorders. The novelty of the original research paper by Salice and Henriksen, in their Hypothesis and Theory Article, aimed to differentiate the sources of social difficulties in schizophrenia spectrum disorder and severe autism spectrum disorder. They proposed a distinction between two kinds of shared intentionality—joint- and we-intentionality—and argue that we-intentionality may be affected in schizophrenia, whereas both joint- and we-intentionality are impaired in autism. They argue that the qualitatively distinct social difficulties are linked to the disorders' different psychopathological cores. Trait-like self-disorders may affect the psychological preconditions for we-intentionality, whereas the psychological preconditions for both forms of shared intentionality are impeded by problems with the ability to “be moved” by others' intentions, perspective-taking, and mind-reading in autism.Consistently, through the novel theoretical approach of shared intentionality, Palumbo et al. proposed a novel scale, viz. the Autism Rating Scale (ARS), to detect and measure the phenomenon of schizophrenic autism. Their article explored the psychometric properties of the ARS and furthermore found that scorings on the ARS differentiated patients with schizophrenia and bipolar disorder.The complex relationship between formal thought disorders, self-disorders, negative symptoms, and social cognition may be reflected in a specific phenotype, which, in part, concerns dis-sociality and detachment from the common sensical world. This phenotype is reflected in the classical phenomenon of schizophrenic autism, which Bleuler also originally described as a fundamental symptom of schizophrenia. In their original research article, Guloksuz and van Os provocatively restate their belief in the death of schizophrenia concept and in the promise of the wider psychosis spectrum concept. They summarize shortcomings to the schizophrenia concept  and highlight the benefits of the psychosis spectrum concept in conjunction with clinical characterization.Finally, in their opinion article, Overall, we hope that this Research Topic dedicated to the nosological promise and perils of the Schizophrenia Spectrum concept will contribute to further advancements in the field.AC, MH, and AR contributed equally to drafting the Editorial and revising it critically. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.