Blinkhorn et al.; Scientific Reports 10.1038/s41598-021-82257-6 (2021).1 present a reanalysis of fossil and lithic material from Garrod’s 1928 excavation at Shukbah Cave, identifying the presence of Nubian Levallois cores and points in direct association with a Neanderthal molar. The authors argue that this demonstrates the Nubian reduction strategy forms a part of the wider Middle Palaeolithic lithic repertoire, therefore its role as a cultural marker for Homo sapiens population movements is invalid.Blinkhorn et al.might have made Nubian technology elsewhere is unsubstantiated.We raise the following four major concerns: (1) we question the assumptions made by the authors about the integrity and homogeneity of the Layer D assemblage and (2) the implications of this for the association of the Neanderthal tooth with any specific component of the assemblage, (3) we challenge the authors’ attribution of lithic material to Nubian Levallois technology according to its strict definition, and (4) we argue that the comparative data presented derive from a biased sample of sites. These points critically undermine the article’s conclusion that Shukbah’s Neanderthals made Nubian cores and thus the argument that Neanderthals 3. In the context of a Palaeolithic cave, a deposit this thick inevitably combines multiple occupation phases that were excavated as a single unit; thus, it is problematic to treat the assemblage as a homogeneous entity. Renewed excavations at multiple key sites in Israel have highlighted the need for caution when referring to stratigraphy described in early twentieth century excavations; for example, at Tabun Cave5 following Garrod's6 original excavation, and at Kebara Cave8 after Turville-Petre9. At both sites, thick and extensive Middle Palaeolithic strata were identified originally but subsequent investigations have shown that these units were in fact composed of many archaeological layers spanning a broad time-range. Since Garrod used a similar field methodology and parameters for defining the stratigraphic units at both Shukbah and Tabun, it is safe to regard Shukbah Layer D as similarly conflating multiple archaeological layers. Other studies of ‘old’ collections have also indicated the problems of stratigraphy from these early excavations and the consequent contextual uncertainties of the lithic assemblages11. We argue that these problems also apply to Shukbah, thereby undermining Blinkhorn et al.’s assumption that Layer D represents a single, archaeologically meaningful assemblage.The Layer D lithic assemblage derives from a brecciated deposit ranging in thickness from 0.2 to 2.5 m, with Layer D material said to be redeposited as disturbed Layer C1. SI7]. While the majority of the assemblage in the Rockefeller Museum collection represents the Middle Palaeolithic, artefacts from earlier and later periods are also evident  is dominated by the centripetal Levallois method Fig. . Bidirec1 is unwarranted.In sum, the mixed collection from Shukbah Layer D in the Rockefeller Museum calls into question the integrity of Layer D as a whole. Furthermore, since technological and typological characteristics from different chronological stages within the Middle Palaeolithic are also present, ascribing unprovenanced sub-samples of the entire assemblage to the late Middle Palaeolithic2 describes that the Neanderthal molar was “found lying upon the rock at the base of the breccia, at a point where it was extremely hard throughout”, specifying that this was “the base of the hummock on the edge of the pit”3. Based on this description and the illustrated excavation section3, the molar derives from the contact between Layer D and the sediments of Layer B, giving it an insecure stratigraphic attribution. Garrod herself questioned the association of other human remains found within the Layer D breccia2, also described elsewhere16.Given the above, it is neither possible to define the stratigraphic component of Layer D that is associated with the Neanderthal tooth, nor to assert its association with the ‘Nubian’ artefacts in the assemblage such as they are (see below). Garrod19. The definition offered in Blinkhorn et al. [ref.1 Methods] states that “Nubian Levallois points and cores have been differentiated from other Levallois point reduction approaches by the presence of a steep medial-distal ridge produced through a combination of distal divergent or lateral removals which help to guide the preferential flake removal”. They cite Usik et al.’s20 suggested set of attributes against which an identification of Nubian cores can be tested and differentiated from other Levallois core types. In this definition, the reduction strategy results in: (1) a steeply angled median distal ridge < 120° and generally > 60°; (2) an opposed striking platform with angle of intersection to the exploitation surface varying from 50° to 90°; (3) a triangular or sub-triangular core shape; and (4) a faceted primary striking platform. These criteria represent the culmination of a methodological consensus from several teams working in different contextual areas23. These definitive criteria have been followed in numerous subsequent studies27. Blinkhorn et al.1 neither follow these criteria, nor present data to fully evaluate the presence of Nubian technology in Shukbah Layer D.Nubian Levallois technology is regarded as a distinct method of Levallois reduction; Levallois cores with distal and lateral preparation are not necessarily Nubian cores and can equally be attributed to the Levallois bidirectional and centripetal modes of core preparation1 SI7: 20]. With the limited information provided, supported solely by illustrations of eight cores [ref.1 Fig. 5a-h], most of which are shown only in plan view, it is impossible to determine whether the cores considered to be ‘Nubian’ comply with the criteria outlined above and, thus, whether they can be differentiated from Levallois bidirectional or centripetal cores. The assemblage also contains 13 “other” Levallois point cores, yet no further details are given with regard to their exploitation strategy and morphological attributes.They do not define the steepness of the median distal ridge on Nubian cores and there are no quantitative or qualitative attributes provided to support this. They identify 16 cores as Nubian, three showing proximal and distal divergent shaping, and 13 with orthogonal or centripetal removals [ref.1 Fig. 5j-n], they incorrectly presume that there is a clear definition of Nubian end-products offered elsewhere20. In the absence of direct refits, Usik et al. do not consider Nubian points to be distinguishable from the end-products of centripetal Levallois reduction; they observe only that “the Nubian Levallois method is based on the production of elongated Levallois points or pointed flakes”20. Based on extensive refitting, Van Peer18 describes Nubian end-products as pointed flakes produced following the central guiding ridge in the distal core part combined with a radial pattern of preparation in the proximal part. At Nazlet Khater 1 in the Nile Valley, Nubian end-products have more dorsal scars than classical Levallois end-products  and tend to be longer, less elongated and thicker28. Given this lack of consensus, it is unclear how Blinkhorn et al.1 separated Nubian end-products from other Levallois end-products (points) for their statistical treatment. They identify nine ‘Nubian’ points with bidirectional dorsal scar patterns and three with a combination of distal and lateral removals [ref.1 SI7: 19]. However, “other Levallois points” include 49 with bidirectional scar patterns, alongside unidirectional, unidirectional convergent and centripetal removals [ref.1 SI Table 22]. The multivariate analyses are said to indicate that Nubian Levallois points are not distinct from the wider body of Levallois points at the site , the attributes upon which the multivariate analyses are based in this paper are solely basic linear measurements and derived indices [ref.1 Methods]. These attributes have been shown to be highly dependent on quality and size of raw material, independent of reduction intensity20 but these aspects are not reported on by Blinkhorn et al.1. Indeed, it is doubtful that they can be, given the biased collection and outdated excavation practices.Although reference is made to metric and categorial traits listed in previous studies by the authors . Given that the analysed artefacts are not shown to fulfil the criteria for Nubian Levallois technology and instead represent other Levallois methods, the conclusion that “Nubian Levallois reduction strategies form part of a wider Levallois point production strategies, rather than a discrete technological approach” [ref.1 SI7: 20] is contained in the methodological premises and, thus, inevitable.No appropriate technological comparisons are made between different Levallois reduction strategies at Shukbah , that justify the grouping into Nubian and Other Levallois . Put simply, the multivariate analyses do no more than indicate the extent of inter-assemblage variability which is expected given the broad spatial, temporal and contextual range of assemblages compared.The choice of comparative assemblages is not well explained and sampling procedures are not justified. Problems include a reliance on very small samples (e.g. < 1% of the Rosh Ein Mor assemblageHomo sapiens and Nubian Levallois technology remains to be demonstrated” [ref.1 SI3: 8], when the available data are properly evaluated, the same can also be said for its association with Neanderthals. Blinkhorn et al. do not demonstrate any association between Nubian technology and Neanderthals at Shukbah Cave. The arguments we present question the integrity of Shukbah Layer D, finding no convincing evidence for associating the Neanderthal molar with any specific artefact types within the mixed assemblage, as shown by our review of the Rockefeller Museum collection. Furthermore, the identification of so-called Nubian cores and points is insufficiently supported by the results presented and these do not explicitly demonstrate the strict criteria accepted by scholars in other recent studies, nor do they present new ones. Lastly, the comparative analyses used to contextualise Shukbah reflect biased samples that are not informative without more detailed consideration of context, raw material and other relevant technological variables.Just as the authors argue that “any association between As our knowledge of the timing and distribution of Nubian Levallois technology comes into focus, it is becoming increasingly clear that this reduction strategy occupied a continuous landscape – a contextual area spanning Northeast Africa, the Arabian Peninsula, and southern Levant – coinciding with a critical stage of modern human emergence. Blinkhorn et al.’s paper demonstrates one essential point: a unified definition of Nubian technology is fundamental to a better understanding of its significance in the processes of adaptation, cultural convergence and dispersal by modern humans in this region during the Late Pleistocene.


Microneedles (MNs) represent the concept of attractive, minimally invasive puncture devices of micron-sized dimensions that penetrate the skin painlessly and thus facilitate the transdermal administration of a wide range of active substances. MNs have been manufactured by a variety of production technologies, from a range of materials, but most of these manufacturing methods are time-consuming and expensive for screening new designs and making any modifications. Additive manufacturing (AM) has become one of the most revolutionary tools in the pharmaceutical field, with its unique ability to manufacture personalized dosage forms and patient-specific medical devices such as MNs. This review aims to summarize various 3D printing technologies that can produce MNs from digital models in a single step, including a survey on their benefits and drawbacks. In addition, this paper highlights current research in the field of 3D printed MN-assisted transdermal drug delivery systems and analyzes parameters affecting the mechanical properties of 3D printed MNs. The current regulatory framework associated with 3D printed MNs as well as different methods for the analysis and evaluation of 3D printed MN properties are outlined. Since the approval of the first transdermal patch containing scopolamine for the treatment of motion sickness four decades ago , the traMicroneedles (MNs) represent skin-friendly puncturing devices of microscale dimensions  that areTo meet all requirements for the non-invasive application and efficient drug delivery to the systemic circulation, a successful MN system must be produced by a manufacturing method that guarantees accurate, reproducible, robust, and precise production of MN in the micrometer range ,10. To dthe fabrication of objects through the deposition of a material using a print head, nozzle, or another printer technology” ..122].In addition to the research interest in 3D printing of solid MNs, researchers also introduced the use of 3D printers to fabricate hollow MNs. A custom 3D printed hollow MN biodevice b consistYeung et al. introduced an interesting approach for microchannel-MN platform 3D printing. They used SLA 3DP to manufacture a single-piece, multi-inlet, 3D microfluidic device with an embedded hollow MN array. This device, consisting of a hydrodynamic mixing module that allows homogeneous mixing of multiple fluids under different flow rates, is interfaced with a hollow MN array able to transdermally deliver mixed drug solutions and facilitating programmable new TDD applications .Recently, Economidou et al. also demonstrated that SLA 3DP can be a valuable tool for the fast and cost-effective production of hollow MN patches. For the first time, this research group presented a universal TDD device consisting of a hollow MN patch integrated with a microelectromechanical system (MEMS). SLA 3DP hollow MNs were paired with a diaphragmatic micropump as a MEMS, to create a 3D printed MN-mediated drug delivery system that enables personalization of the treatment through in situ control of drug administration by the user. In vivo results showed that administering insulin with this innovative device improved glycemic control in diabetic mice compared to subcutaneous injections .Although SLA technology can be very successfully used to manufacture MN arrays and MN-mediated TDDs, the successful commercialization of such products will require numerous additional investigations regarding optimization of printing and post-printing parameters and selection of appropriate biocompatible and safe printing material.Economidou et al. recently made an important contribution in this context by investigating the effects of printing angle and post-printing curing conditions on the sharpness and mechanical properties of SLA-printed MNs. It was found that the post-printing curing conditions influenced the mechanical properties of the material and MNs due to the degree of cross-linking. These results suggest that optimizing the post-printing curing regime is a crucial step for the photopolymerization of printed MNs. The printing angle proved to be very important as it also influenced the MN quality and dimensional accuracy, where the print quality at 45° seemed to improve significantly and the MN appeared sharper without any structural manufacturing faults being detected. Results also suggested that MN geometry and geometrical parameters influenced piercing force and coating morphology and dissolution, respectively .Digital light processing (DLP) is photopolymerization-based technology that differs from SLA only in the light source used . DLP is Miller et al. created a hollow MN array out of a photosensitive acrylate-based polymer resin using DMD™-based microstereolithography . Gittard® AN 169 BF . MNs were then loaded with miconazole using a piezoelectric inkjet printer ..104].Printing accuracy and light intensity are the main differences between DLP and LCD 3D printing, where the intensity of LCD 3D printing is very weak, and the precision of LCD printing technology is inferior to DLP. Nevertheless, LCD 3DP offers satisfactory resolution down to 150 μm in the horizontal direction and 50 μm in the vertical direction, enabling the low-cost production of microstructures with complex architectures .Recently, Xenikakis et al. presented for the first time the fabrication of a hollow MN device consisting of an array and a reservoir a by the In 2015, Carbon 3D Corp  developed new continuous liquid interface production (CLIP) technology as a novel alternative to traditional layer-by-layer SLA . The invOnly one year after its introduction, the CLIP technology was successfully used to rapidly prototype MNs for TDD. Johnson et al. utilized the CLIP technique to rapidly manufacture sharp MNs with different geometries in one step b. These Free radical photopolymerization, which occurs during the CLIP process, has the potential to damage encapsulated therapeutics, particularly sensitive biologics. On the other hand, the stability of the API incorporated in dissolvable, degradable, or swellable polymer MNs must be maintained during the MN fabrication process. To avoid possible destabilization of encapsulated cargo during the CLIP process, Caudill et al. proposed to coat therapeutic agents on the surface of CLIP-assembled MNs . In theiAs the most accurate 3DP technology to date, the two-photon polymerization (TPP) or 2PP technique enables layer-by-layer fabrication of 3D structures from solid, liquid, or powder precursors for microscale and nanoscale structures. Induced by a near-infrared femtosecond laser, TPP can fabricate arbitrary and ultraprecise 3D microstructures with high resolution  . The tecThis fast and one-step process offers several advantages over conventional manufacturing techniques for scalable mass production of MNs. TPP can easily be scaled up for industrial use, there is no need for cleanroom facilities, and it can use various low-cost materials such as ceramics, polymers, and other photosensitive materials. Many research groups have exploited this technique to fabricate solid ,109,110 ® . Obtained MNs exhibited appropriate mechanical properties, and specific penetration without fracture, against porcine skin surfaces [® MNs with a larger range of geometries than conventional microfabrication techniques [Doraiswamy et al. were the first to report using TPP to produce MNs from non-toxic and biologically inert Ormocersurfaces . Ovsianichniques . Gittardchniques . Anotherchniques .Hollow MNs combined with internal laser-generated microchannels were developed by Trautmann et al. using TPP technology. This novel hybrid approach combining TPP-printed MNs with femtosecond laser-generated microfluidic channels provides an important step towards versatile medical point-of-care systems . Moussi ®) was introduced as an innovative high-precision AM technology that combines the extraordinary performance of grayscale lithography with the precision and flexibility of TPP technology. This interesting technology is very accurate and ultra-fast, offering very high spatial resolution in the submicron range. Indeed, the minimum lateral size of the printed voxel can be as small as a few hundred nanometers. We believe that this maskless lithography technique will contribute to a new era of high-precision manufacturing of MN arrays [In 2019, two-photon grayscale lithography , selective laser melting (SLM), and direct metal laser sintering (DLMS). These techniques require a high energy flow on the powder bed of building material to produce the desired objects , while tSelective laser sintering (SLS) is powder bed 3D printing technology invented by Carl R. Deckard for his master’s thesis at the University of Texas and was patented in 1989 (US 4863538 patent) ,153. ThiDMLS can be used to produce small, geometrically complex, fully functional metal parts such as MNs from medical-grade materials with great freedom of design, which would be difficult to produce with classical methods ,156. TheSun et al.  investigKrieger et al.  used a DThe working principle of SLM is similar to SLS and DMLS in that the powder is spread by a roller on the building platform and a laser fuses the powder on points defined by CAD design data. Subsequently, the platform is lowered and another layer of powder is applied . In contTo produce hollow MNs from stainless steel 316 L, Gieske et al.  modifiedinsertion into the skin at sufficient depth without breaking,dimension and design with optimal properties, andusing proper (biocompatible) material.In order to be considered a successful drug delivery system, the MN array patch must meet several criteria, such as ,161:inseSince various methods of 3D printing of MNs have been developed, it is necessary to carry out optimization studies on the design and dimensions of MNs, but considering the 3D printing quality, which strongly depends on the set printing parameters . HoweverIt is well known that materials used for MN manufacturing must have sufficient strength to penetrate biological barriers, must be easily manufactured, and be compatible with drug molecules. Material properties affect critical parameters such as stability, tensile strength, drug loading, and biocompatibility of MNs ,163. BioAll photocuring 3DP techniques require the printed materials to have a photosensitive property and to be composed of precursors, photoinitiators, adsorbers, fillers, and additives . Most of®) [Methacrylate-based resins are the most commonly used materials in SLA manufacturing of MNs ,126,127.®) ,147 or a®) ,148.Mansor et al. investigated several commonly used polymers as candidates for the SLA process. They prepared solid MNs using PVA, PLA, polyester resin, and ABS and characterized their mechanical properties. Results showed that PVA had had the highest ability to withstand force over other applied materials .Standard materials used in FDM printers are thermoplastic filaments such as acrylonitrile butadiene styrene (ABS), PLA, PVA, high-impact polystyrene (HIPS), polyethylene terephthalate glycol-modified (PET-G), and nylon. Biocompatible PLA was predominantly used in FDM MN fabrication . While SThe choice of materials should be carefully considered to enable safe and effective MN devices to be delivered to patients . ImproveIn order to achieve the appropriate product quality, a detailed analysis of the impact of all important process parameters is required, regardless of the 3D printing method. Optimization of process parameters is a key criterion for achieving the appropriate product quality in terms of improving dimensional and geometrical accuracy and surface quality .For MN production, printer resolution is one of the most important parameters for achieving the desired MN design and forming sharp MN tips. Print resolution is influenced by the software and hardware components of the device. Of the software components, the greatest influence on the quality of the product itself is the mesh density when forming the .stl format ,170,171.Layer thickness is one of the process parameters that has the greatest influence on the printing resolution in the z-axis direction (build direction). This influence is especially significant in the building of curved surfaces due to the pronounced “staircase” phenomenon. As the layer thickness increases, the accuracy of the overall finished sample geometry decreases, and the “staircase” phenomenon is more pronounced  ,173.The length of MNs can usually vary between 150 and 2000 μm and guarantees a direct penetration of MNs through the epidermis layer to the capillary system of the skin in dermal tissue . The lenThe radius of the curvature is crucial for efficient MN insertion. If the MNs are blunt or too short, the skin may fold around them during application and prevent their penetration ,128. An The significance of MN tip geometry for MN insertion was studied by Davis et al. They measured the force required to insert MNs into living skin and the force that MNs can withstand before fracturing. The insertion force varied linearly with the interfacial area of the MN tip. Increased wall thickness, wall angle, and tip radius resulted in a higher force required for MN fracture. Reduced MN tip radius from 80 to 30 µm improved insertion efficacy because the smaller tip required the lower insertion force to penetrate through the SC . A low tThe density of the MN arrays is also important for efficient skin penetration ,128. It The 3D printed MNs may also vary depending on their tip or overall shape, as shown in The laser-based writing systems are capable of printing MN arrays of the same design with high degrees of consistency . TherefoThe shape of MNs influences the force required for successful penetration into the skin. Pere et al. concluded that the conical MNs require the least force to penetrate porcine skin compared to pyramid geometries, probably due to the difference in the MN-to-skin contact surface between these two designs . EconomiYeung et al. also employed SLA to produce hollow MNs with conical, pyramidal, and fine-tip syringe-shaped designs. They note that pyramidal MNs only left marks on the first layer of parafilm, while the conical ones merely indented the first layer of parafilm. However, the best results were obtained with the syringe-shaped MNs, which left significant imprints on the second layer .It is noteworthy that for optimal results and obtaining MNs with suitable quality and performance, in addition to the manufacturing parameters of 3D printing, the geometric characteristics of MNs also need to be optimized. Manufacturing parameters are notoriously sensitive to 3D printing efficiency, and an application as challenging as MNs necessitates a deep understanding of the relationships between parameters and quality features . Table 2Geometry, dimensions, surface morphology, and distribution of MNs on the array can be determined and evaluated by visual inspection, stereomicroscopy, and optical or scanning electron microscopy . PropertFluorescent labeling or dyeing the molecules incorporated in the MN patch can be used for their identification. Successfully incorporated molecules may be visualized by confocal laser scanning microscopy (CLSM), fluorescent microscopy, or even visual inspection. Visualization is useful for the localization of molecules incorporated within an MN patch whether it is the tip, shaft, or the backing layer . Coated MNs are exposed to a variety of stresses during insertion due to non-uniformity of the skin surface, unavoidable movements, and stresses exerted upon removal . MechaniThe significance of these tests is to determine whether the MNs have sufficient mechanical strength to withstand deformations and any other undesirable changes during handling and skin insertion.This type of test involves measuring the failure of MNs caused by axial or transverse loading. The test station presses the MN array parallel against a rigid metal surface followed by the measurement of force and displacement while generating the stress against strain curves a. If MNsTransverse fracture force tests are important for the assessment of the application of MNs, and they can be applied to a single MN, and a row of MNs as well, for which the force should be divided by the number of MNs in a row to calculate the transverse fracture force per individual MN. The main limitation of this test is the required manual alignment of the metal probe with a defined length, which can cause inaccuracies . FailureThe fracture of the baseplate during application by the patient is not acceptable, so its strength should also be determined. The flexibility of the MN baseplate ensures appropriate insertion on the skin surface. The degree of flexibility should be sufficient to conform to the irregular topography of the skin without fracturing. To do this, a bending test with three points can be used. Baseplates are placed between two aluminum blocks and the force is applied by a metal probe. The force required to break the baseplate can be measured by observing a maximum peak value on the force–distance curve. The flexibility of the baseplate can be calculated from its bending upon fracture .Knowledge of the insertion capability of MNs enables the prediction of the appropriate MN length required for their insertion into the skin due to the inherent viscoelasticity of the skin. Measuring the insertion force required to pierce the skin by MNs is also important to ensure a complete and equivalent assessment of fracture forces. Fracture forces should be significantly higher than the insertion forces required for inserting MNs into the skin. MNs insertion can be done manually or by using different applicators b. ApplicDue to the inherent viscoelastic properties of the skin, it is difficult to achieve complete insertion of the whole MN length. The depth of MN insertion can be divided into two categories: true depth and estimated depth. Methods that provide true depth include confocal microscopy, X-ray transmission computational tomography (XRTCT), and optical coherence tomography (OCT). The estimated depth of MN insertion can be evaluated by histological cross-sectioning and staining of the skin by colored dyes after MN application. The most commonly used dyes are gentian violet, methylene blue, and trypan blue. These dyes enable the visualization of microchannels generated by MNs in the epidermis. Only the cells of the viable epidermis are colored, while the cells in the SC remain as they were before the MN application. Furthermore, the penetration success ratio of MNs may be calculated as a relation between the number of dye-stained spots observed and the total number of MNs per array . The limHistological cryosectioning implies removing the skin treated with MNs from the bulk skin sample, fixing it in a suitable medium, and then immediately freezing it by using liquid nitrogen and storing it at −80 °C. The thickness of cryosections is between 6 and 12 µm and they are usually stained with hematoxylin and eosin to observe the microchannels created by MNs . This inCLSM is an adequate and non-invasive method for measuring the dimension of pores created by inserted MNs. It is based on the exposure of the treated skin area to a solution with specific fluorescent microparticles that migrate down the channels created by MNs. Afterward, these fluorescence probes are detected by CLSM and they indicate the depth of the pores. The penetration depth of CLSM is only 200–250 µm from the skin surface, which limits the possibility of using this method for measuring microchannels made by longer MNs ,194. AnoOCT is a good method for evaluating the effects of MNs’ geometry on skin penetration and in-skin dissolution. This non-invasive method allows the determination of the actual depth of MN insertion into the skin in real time. There is no skin excision or mechanical manipulation of samples. It can visualize depths of ~2000 µm ,196. It ® , a blend of hydrocarbon wax and polyolefin, which can be used as a skin model to study the insertion properties of MNs. Parafilm M® simulates the elasticity and mechanical properties of the skin and allows a quick and rapid assessment of the insertion depth of different MNs.XRTCT is another non-invasive method that uses a series of X-ray scans taken at different rotation angles that generate 3D volumetric data, which allows 3D visualization of the MNs’ insertion. It provides the ability to see if all MNs in the array were able to breach and penetrate the skin or if there were regions within the patch that could not penetrate the skin due to mechanical failure. The main limitation of this method is that only MNs made of material with X-ray contrast properties, such as gold, can be observed. It also does not sufficiently distinguish the exact skin layers penetrated by MNs ,200. To Insertion efficacy can also be evaluated by measuring the transepidermal water loss (TEWL) or the electrical impedance (EI). Both methods are based on the reduction of the barrier function of the SC but cannot provide quantitative information about the insertion depth of MNs ,202. MeaDepending on the size, material, and the type of the delivered drug, mild and transient erythema may occur as a side effect after MN application. The irritation can be observed dermatoscopically or stereomicroscopically. The degree of skin irritation, although subjective and variable, can be determined by the Draize method in which the skin is observed macroscopically before and after the application of MNs and dermatological changes are evaluated according to the degree of erythema and edema . Skin reThe amount of the drug delivered into the skin can be determined by diffusion cells. Usually, the test is performed on ex vivo human or animal skin or an artificial polymeric membrane placed between receptor and donor compartments. The observed permeant is inserted into the donor compartment, which diffuses into the receptor compartment via the membrane of choice . TransdeThere are two types of diffusion cells: static (Franz type) and flow-through (Bronaugh type). Franz-type diffusion cells can be divided into side-by-side or upright cells, depending on the orientation of the skin in the diffusion cell. Side-by-side static diffusion cells are seldom used in studies of skin permeation nowadays due to skin damage caused by complete immersion of both SC and dermis in donor solution and receptor media and stirring the solutions at the same time. An overestimated penetration profile can also occur due to excessive and prolonged hydration of the SC through the donor compartment. An upright diffusion cell implies clamping the skin between a receptor compartment containing the receptor media while the SC side faces the open donor compartment, imitating the typical environment to which it is usually exposed ,208. WheBronaugh-type, flow-through cells maintain a sink condition by using a peristaltic pump that ensures a continuous flow of receptor media through the receptor chamber. Such continuous flow beneath the skin imitates the dermal circulation that carries the exogenous compound away from the absorption site. Moreover, these cells can use automatic sampling that can overcome labor-intensive sampling time points while allowing continuous monitoring of the absorption profiles . DespiteNeither in vitro nor ex vivo models can fully replicate the in vivo conditions and all skin characteristics. In vivo studies are necessary to investigate the absorption, disposition, and permeation of drugs delivered by MNs. Thickness and elasticity of the skin are crucial parameters that must be considered while selecting the appropriate in vivo model. Although there are differences in structural, histological, and morphological characteristics compared to human skin, the pig model is considered to be the most suitable for transdermal delivery studies . RodentsEven though 3DP TDD systems offer a variety of possibilities for a suitable approach to every individual patient, regulatory requirements for their application are still not precisely defined. MN patches are considered as devices and have to abide by the terms of good manufacturing practice (cGMP), described in the Quality System Regulations (QSR) ,217. AM Quality requirements of the final product, its safe and effective use, biocompatibility, sterilization, and validation of design must be fulfilled before they can be commercially available. The FDA requires the examination of manufacturing process information. Detailed guidelines are given in their published document “Technical Considerations for Additive Manufactured Medical Devices” . InformaThe unprecedented ease with which complex objects are produced and the relatively low price of commercially available 3D printers have given 3D printing significant popularity and has been spoken of as a third industrial revolution. It has the inherent ability to revolutionize conventional pharmaceutical manufacturing by adjusting the size, shape, and release profile of different drug delivery systems, allowing the precise preparation of personalized dosage forms to address individual patient needs. This technology can play a very important role in the manufacture of personalized TDD systems, as evidenced by the growing interest in the 3D printing of MNs. MNs represent next-generation therapeutic systems that may have a notable impact on clinical medicine in the future, but further multidisciplinary research is needed to obtain ideal MN-based point-of-care systems. Having in mind that AM can be used to rapidly prototype different MN designs as well as MN-based TDD, this approach will undoubtedly optimize the effectiveness of these delivery systems and open new horizons for researchers in the field. Even though photopolymerization-based technologies have been the most widely used 3DP technology in the production of MNs, there is also great potential in all other technologies. However, clinical translation and commercial development of 3DP MNs are still a challenge, and a lot of future work is needed, especially in material selection, optimization of printing and post-printing parameters, and drug loading approaches.Further investigations are needed to find relationships between MN design and printing parameters and their quality and performance. In addition, future studies need to focus on improving printer properties, such as the laser beam in laser-based methods or nozzle features in extrusion-based methods, to develop faster methods with the highest resolution. Standardized evaluation methods and testing protocols for 3DP MNs also need to be further developed as the variety of mechanical equipment used gives divergent results that are sometimes very difficult to compare. Advances in 3D printing techniques for MN production, as well as recent breakthroughs in electronic mechanicals and artificial intelligence, offer enormous potential for the development of TDD systems that would allow patients to self-administer drugs such as vaccines.


For example, results of computations may be taken at face value as “nothing but the truth” and data visualization may be used only to generate “pretty and colorful pictures”. Computational experts might come to the rescue and help to re-direct such efforts, for example, by guiding interested novices to conduct meaningful data analysis, make scientifically sound predictions, and communicate the findings in a rigorous manner. However, this is not always ensured. This contribution aims to encourage investigators entering the CADD arena to obtain adequate computational training, communicate or collaborate with experts, and become aware of the fundamentals of computational methods and their given limitations, beyond the hype. By its very nature, thisOpinion is partly subjective and we do not attempt to provide a comprehensive guide to the best practices of CADD; instead, we wish to stimulate an open discussion within the scientific community and advocate rational rather than fashion-driven use of computational methods. We take advantage of the open peer-review culture ofF1000Research such that reviewers and interested readers may engage in this discussion and obtain credits for their candid personal views and comments. We hope that this open discussion forum will contribute to shaping the future practice of CADD.The current hype associated with machine learning and artificial intelligence often confuses scientists and students and may lead to uncritical or inappropriate applications of computational approaches. Even the field of computer-aided drug design (CADD) is not an exception. The situation is ambivalent. On one hand, more scientists are becoming aware of the benefits of learning from available data and are beginning to derive predictive models before designing experiments. However, on the other hand, easy accessibility of In recent years, software for CADD has become more widely accessible. Today, a range of software packages are available that are open-source or free to use for academic research. Together with significant alterations of the scientific landscape induced by the COVID-19 pandemic and the ensuing re-orientation of some early-career but also established research groups towards the use of computational tools, this has boosted the use of CADD methods in particular in academic research environments. However, low-barrier access to computational tools and computing power increasingly leads to the use of CADD techniques also by scientists who have not received formal training on these methods. Many newcomers to CADD employ easy-to-use software without realizing the complexities involved and without being aware of the many potential pitfalls. Improper use of CADD techniques can have a contrary effect on research than intended. The risk of generating meaningless or false predictions is high. Flawed predictions can lead to dedicating significant resources to futile experiments. In particular, publishing invalid predictions can lead to error propagation, eventually resulting in a loss of confidence in CADD. Moreover, uncritical or naive use of artificial intelligence (AI) methods that are being heavily promoted in many research fields has similar negative effects, working against the credibility and acceptance of CADD as a scientific discipline.Computer-aided drug discovery (CADD) has become a key technology in drug discovery, providing guidance to experimentalists on which compounds and experiments to focus on next. The capacity of CADD has further increased by the development of powerful machine learning approaches, deep learning in particular. Similarly, best practices in different stages involved in CADD have been discussed, for instance, in quantitative structure-activity relationship (QSAR) analysis, data curation, molecular docking and virtual screening.Good practices to conduct and report studies in computational medicinal chemistry and chemoinformatics have been outlined in several articles.In this contribution, we discuss common misconceptions and false expectations associated with CADD, especially in the AI era, and make recommendations on how to avoid common pitfalls when using CADD software. We aim to stimulate an open discussion within the community to help improve our perception and practice of CADD and contribute to shaping its future. Importantly, theoretical disciplines are an integral part of CADD, establishing its scientific foundations. The ability to apply such approaches using software does by no means guarantee that reasonable research is carried out. Therefore, any conclusions or claims should be carefully considered.Experts from various disciplines involved in drug discovery, such as chemical synthesis and biochemistry, are increasingly making use of computational tools to guide their experimental research and rationalize their observations. This is a positive trend, as developers of CADD tools have been aiming for a long time to make their software more widely accessible and intuitive to use. Prominent examples include web servers, and commercial, free and open-source software. But to develop further, it is of paramount importance to avoid confusion about the concepts and differences between the different areas in drug discovery such as molecular modeling, chemoinformatics, and theoretical chemistry. Conceptual and practical differences between these disciplines are clearly described in the literature.The advent of more advanced computational tools, many of which are open source, freely accessible, and promoted as “easy-to-use,” also increases the widespread use of “buzz words,” and misconceptions among newcomers to CADD.Traditionally, there is a widespread belief in experimental sciences that experimental results represent reality, disregarding the different way in which natural phenomena can be represented and perceived and the relativity associated with varying representations. This ideological attitude works against “out of the box”, hinders intellectual progress, and indirectly de-values scientific disciplines such as CADD. With the rise of AI as one of the most heavily promoted approaches in contemporary society, the academic community has been encouraged to redirect its attention to computational tools to enhance its research impact and appeal. Nevertheless, unconditional trust in “experimental reality” reduces CADD to a “tool provider” and does not regard it as an independent scientific discipline in its own right. Consequently, computational models are often used without the necessary theoretical understanding and the rigor needed to apply them systematically.In the authors’ opinion, one of the first requirements a new computational practitioner needs to address is realizing that both experimental and computational results are constrained by the model or experimental framework applied to determine them and, in no case, an absolute account of reality. Among medicinal chemists, there is the frequent misconception that purely theoretical or computational studies are in principle “incomplete” because there are no “real” experiments. However, such views require reconsideration and correction, as pointed out above. Rigorous computational studies answer questions that are difficult to address without “in silico experiments”. As such, they are comprehensive and self-contained, regardless of whether the computational approach has led to experiments. “Complete” computational investigations are often consistent with prior experimental observations, but may also chart new scientific territory. Of course, new computational insights leading to experimental work trigger interdisciplinary research. This is a noted strength of CADD, if conducted properly. However, there are misconceptions at interfaces between computation and experiment. For instance, a common malpractice is trying to replace enzymatic inhibition assays with predictions based on molecular docking or dynamics simulations. Another misunderstanding is that black box predictions from machine learning would represent a form of “alchemy”. What we cannot understand is not necessarily incorrect and may have value. The catch is that we are left with making decisions in such cases, for example, about new experiments that go beyond our reasoning and hence require trust in computational work and prior experience. It is also false to believe that AI in its current state would provide solutions to questions that replace our judgment capacity. Data volumes quickly go beyond our comprehension but results of statistical analysis of pattern recognition do not replace human reasoning . Furthermore, there is a severe misconception that computational predictions might demonstrate or “validate” the bioactivity of compounds. Notably, these and other misunderstandings may not be evident to researchers and students who are just beginning to use computational methods. We encourage the community to avoid judging a computational research project to be “incomplete” because it does not include experiments or to be “complete” just because it incorporates many different computational methods. The question of completeness is not separable from scientific rigor and adequate conduct of methodologies, be they computational or experimental in nature. Furthermore, let us not consider a computational analysis as a “luxury item” to decorate a project report, grant application, or scientific paper with “pretty pictures”.Mainstream media usually disseminate inaccurate or exaggerated reports about the capabilities of computational methods without also mentioning their limitations and flaws. Simultaneously, mass job search engines commonly offer job opportunities with extensive lists of different computational tools as requirements. These factors, among others, continuously put pressure on researchers to increase their productivity and academic credentials to further their careers at the expense of scientific rigor and the quality of research. CADD is not the exception of the increasing trend that disrupts the traditional academic structure in favor of a more market-oriented approach. One of the consequences of this phenomenon is that many young professionals and new CADD practitioners direct their efforts to increase the volume of their curriculum vitae rather than using CADD methods to answer relevant scientific questions. which is often confused with assessing “drug-likeness”. Another common pitfall among newcomers to CADD is using docking to predict “real” protein-ligand complexes, given its popularity and easy-of-use. Practitioners should use methods for the right reasons and not just because everybody else is using them. This requires knowledge of underlying theories and sound scientific judgment.The popularity of computational methods or tools often jeopardizes rational selection. Newcomers often turn to frequently used methods that are well-validated. However, the justification is questionable if the technique is merely used because it is “popular” . Without properly addressing the question at hand, computational analysis applying irrelevant approaches is misleading or propagates errors. Arguably, one of the most misused guidelines in drug discovery is the Lipinski Rule of Five,•Intense study of the literature is essential to acquire knowledge. Like experimental techniques, also CADD methods require proper training to become familiar with their applicability domains, approximations and limitations.•Computational research projects should primarily be problem-oriented rather than technique-oriented, unless the development of new techniques themselves is the focus of the problem to be addressed. Projects (including dissertations) should be well-structured according to scientific criteria or milestones, but by no means represent a compilation or aggregated use of techniques applied to the same data. Before deciding which computational approaches and tools to use, a comprehensive research of the literature should be conducted and exemplary applications should be reviewed. Then, based on the experimental information available, appropriate computational methods and strategies should be applied. Rushing into calculations with software packages, even with excitement, is typically detrimental if the applied methods are not scientifically justified. In addition to researching methodological aspects, it is also mandatory to carefully review the available experimental findings. For example, prior to applying virtual screening techniques to search for new compounds with activity against high-profile and extensively explored targets, care should be taken not to overlook prior art in the field and avoid engaging in scientifically naive computational efforts. One should avoid setting the goals of a drug discovery project relative to a technique by pursuing a “tool-oriented approach”. Instead, planning computational components of an interdisciplinary research project should focus on the ultimate scientific goals. Students should realize and keep in mind that learning and applying different techniques across disciplines is desirable, but they should be used in harmony to answer research questions.•Seek supervision or advice from experts and do not hesitate to ask. Consultation prior to engaging in a new scientific adventure will not only save time and resources but also help to plan a scientifically sound approach.•Avoid excessive use of buzzwords such as “artificial intelligence” or “machine learning” when they are not applicable, which contributes to inappropriate hype associated with computational methods. For example, there is no need to use the AI or “machine intelligence” label for compound classification methods that are already applied for decades.•Keep in mind that many theoretical disciplines contribute to CADD, which have a long history on their own such as machine learning.•As in any wet lab experiment, input data quality is of critical importance for the outcome of computational studies. Awareness of data curation requirements is essential for the integrity of computational work.•The uncritical or uneducated use of web-accessible computational tools or servers to generate new compounds, calculate molecular properties, or predict target structures and protein-ligand complexes is a major source of errors propagating through interdisciplinary projects.In the authors’ opinion, the following recommendations should be helpful to CADD novices and multidisciplinary research teams attempting or planning to use computational approaches to guide drug discovery projects. Similar toOpinion, we wish to encourage students, newcomers, and practitioners of CADD to use the computational tools and resources for the right reasons, not just because they are easily accessible. Similarly, we highly encourage the scientific community to avoid applying computational methods just because they are popular. Instead, it is strongly recommended to identify scientific questions that can be addressed appropriately using CADD approaches - and avoid others where computational efforts become questionable. In general, computational studies that cannot be reported in established peer-reviewed journals whose scope includes CADD are to be considered with appropriate caution, both by experts and novices to the field. This also applies to the use of modeling web servers. While the integrity of publicly accessible computational tools can be guaranteed by the developers, addressing ill-defined questions or tasks using these tools is beyond their control. Recognizing the benefits of the open post-publication review culture ofF1000Research, we would be delighted if this contribution would catalyze open discussions among readers to raise further awareness of latent problematic issues in the CADD area and support its further scientific development.The current pandemic and related funding constraints in some countries and institutions have motivated many researchers at different levels to redirect their efforts from difficult to sustain experimental studies to easy-to-use computational tools that can be employed remotely. Also, reviewer panels of many current grant applications from academia, non-for profit, or the industry currently tend to give priority to research proposals that involve AI. Although this contributes to the popularity of CADD, it also comes at a cost. If uneducated CADD studies enter the realm of science fiction harm is done to this field, its credibility and acceptance, and further scientific development. This must be avoided at all costs. The methods used in CADD should not be applied as black boxes, which can be enabled by just a few hours of hands-on experience such as provided in workshops. Without sufficient understanding of the scope, complexity, and theoretical foundations of these computational methodologies such efforts will inevitably fail and discredit investigators and their work as well as the field as a whole. Newcomers to the area, including students, early-career scientists, and seasoned investigators attempting to re-focus their efforts should be fully aware that, similar to experiments, profound knowledge of CADD concepts and informed use of CADD tools is a must. A simple yet fundamentally important rule applies: “Don’t compute what you don’t understand”. In addition to the general recommendations outlined in thisNo data is associated with this article. The authors discuss the dangers of the growing trend of using computer assisted drug discovery tools without a proper training nor understanding of the fundamental concepts laying behind such approaches. The topic is indeed very important to discuss, and the past year of working away from the bench and the tremendous amount of publications produced using, often not totally correctly, CADD and other bioinformatic tools and the examples illustrated in Table 1, emphasize this need of reminding that computational tools, although easy to use, still require understanding of the concepts they are built on. Regarding the sentence ‘Avoid excessive use of buzzwords such as “artificial intelligence” or “machine learning” when they are not applicable, which contributes to inappropriate hype associated with computational methods', I would also add to avoid the usage of “deep learning” another very trendy buzzword, too often misused. I thank the authors for this article, as such topics are extremely important to be voiced out.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This review about fashion and hype in the field of drug design, written by experts, is indeed very timely and of high interest. The observation applies to many fields related to medicine and biology, to most technologies and even research topics. This opinion paper should be read by politicians, decision makers, journalists, students, the citizens not involved in research and many scientists. Even more so, it should be read by people defining guidelines for grant applications and by investors that are ready to put millions on a new “in silico” technology that is going to save the world but that has never been published and is only documented on some nice brochures full of buzz words . Indeed, nowadays, if a grant or a research paper does not use every two sentences words like game-changer, disruptive, AI, big data, multi-scale, large scale, ultra-large something or quantum something,  it has little chance to succeed . The complexity is that, in some cases, these methods are going to help, in some others, this is the wind blowing in the forest. Experts in the field know but they are not invited to comment or, if they are, they do not say anything because they are afraid to be considered old school. The reality is of course very different, no problem being enthusiastic about some approaches, about developing "user-friendly" methods, about testing new concepts, but global brainwashing, overselling and propaganda about AI and related are damaging research and in the field of health, these promises will hurt patients, and confuse even more people. How to stop this given the millions of dollars behind, given global mass brainwashing and the fact that most humans prefer fairy tales than truth? I do not know. Definitively the present opinion paper can help, the one difficulty I see is that people who should read it will not do so and worse, do not want to hear about such discussion as it may kill their business plan or chance of getting famous. Maybe, if the scientific community that refuses “fashion research” starts to make noise, some changes will occur.Is the topic of the opinion article discussed accurately in the context of the current literature?YesAre arguments sufficiently supported by evidence from the published literature?YesAre all factual statements correct and adequately supported by citations?YesAre the conclusions drawn balanced and justified on the basis of the presented arguments?YesReviewer Expertise:drug discovery, structural bioinformatics, chemoinformatics, molecular medicineI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Endogenous cardiac pacemaker function regulates the rate and rhythm of cardiac contraction. The mutation p.Lys23Glu in the cohesin protein Shugoshin-1 causes severe heart arrhythmias due to sinoatrial node dysfunction and a debilitating gastrointestinal motility disorder, collectively termed the Chronic Atrial and Intestinal Dysrhythmia Syndrome, linking Shugoshin-1 and pacemaker activity. Hyperpolarization-activated, cyclic nucleotide-gated cation channel 4 (HCN4) is the predominant pacemaker ion-channel in the adult heart and carries the majority of the “funny” current, which strongly contributes to diastolic depolarization in pacemaker cells. Here, we study the mechanism by which Shugoshin-1 affects cardiac pacing activity with two cell models: neonatal rat ventricular myocytes and Chronic Atrial and Intestinal Dysrhythmia Syndrome patient-specific human induced pluripotent stem cell derived cardiomyocytes. We find that Shugoshin-1 interacts directly with HCN4 to promote and stabilize cardiac pacing. This interaction enhances funny-current by optimizing HCN4 cell-surface expression and function. The clinical p.Lys23Glu mutation leads to an impairment in the interaction between Shugoshin-1 and HCN4, along with depressed funny-current and dysrhythmic activity in induced pluripotent stem cell derived cardiomyocytes derived from Chronic Atrial and Intestinal Dysrhythmia Syndrome patients. Our work reveals a critical non-canonical, cohesin-independent role for Shugoshin-1 in maintaining cardiac automaticity and identifies potential therapeutic avenues for cardiac pacemaking disorders, in particular Chronic Atrial and Intestinal Dysrhythmia Syndrome. A mutation in Shugoshin-1 causes the Chronic Atrial and Intestinal Dysrhythmia (CAID) Syndrome, but the underlying mechanisms are unknown. Here, the authors show that Shugoshin-1 controls cardiac pacemaker activity by interacting with HCN4 to enhance its cell-surface expression, and that the CAID-Syndrome mutation disrupts cardiac pacemaking by interfering with this important non-canonical interaction. During the G1 stage of mitosis, open cohesins are “loaded” onto chromatids via Nipped-B-Like Protein (NIPBL)/MAU2 heterodimers, upon which they close and encircle sister chromatids. Conversely, cohesins are progressively unloaded by Wings Apart-Like homolog WAPL and PDS5, with only the centromere cohesins being maintained until chromosomes are properly aligned2. To prevent premature unloading, cohesins are stabilized by Shugoshin 1 (SGO1), which recruits protein-phosphatase 2 A (PP2A) to antagonize SA-phosphorylation4. SGO1 is removed when phosphorylated by Polo-Like Kinase 1 (PLK1), which leads to cohesin cleavage by separase. Cohesins are also epigenetically involved in transcriptional regulation via Topologically Associating Domains (TADs) and enhancer-promoter loops via interactions with the insulator protein CTCF6. Mutations in cohesin core proteins, NIPBL, ESCO1, and ESCO2 acetyltransferases, or Histone Deacetylase 8 (HDAC8) have all been associated with “cohesinopathies”, in which syndrome-specific changes in cell-cycling and/or transcriptional regulation produce craniofacial, neural and cardiac defects, gastrointestinal dysfunction, developmental delay, or premature ageing6.Cohesins are ring-like structures consisting of four core units: two Structural Maintenance of Chromosomes protein subunits (SMC1 and SMC3); a kleisin subunit (RAD21); and a Stromal Antigen subunit (SA1 or SA2)8. Depletion of SGO1 in HeLa cells results in similar anomalies: premature loss of centromeric cohesin, premature segregation of sister chromatids, and mitotic arrest9. In mice, knockout of both Sgo1 alleles results in embryonic lethality whereas Sgo1+/− mice show a propensity for colon cancer while remaining viable and fertile10. Sgo1+/− mice also show increases in β-amyloid accumulation (of unclear mechanism) in the brain, resembling those seen in Alzheimer’s disease11. We recently identified a series of patients presenting with a combination of heart-rhythm disorders, mainly very slow rhythms due to failure of endogenous cardiac pacemaking function requiring the implantation of artificial pacemakers, and chronic intestinal pseudo-obstruction (CIPO)5. We termed this syndrome, with evidence of both atrial and intestinal dysrhythmia, Chronic Atrial and Intestinal Dysrhythmia (CAID) syndrome. All CAID patients were homozygous for a recessive point mutation (A > G) in SGO1, leading to a lysine-to-glutamic acid change at the highly conserved amino acid 23 (p.Lys23Glut: SGO1-K23E). In contrast to other cohesinopathies, CAID-patients have no craniofacial or structural cardiac defects, nor premature ageing12. CAID-related symptoms appear to mainly, and postnatally, affect the heart and gut, suggesting that the SGO1-K23E mutation likely leads to a change of function with relatively mild, but cell-specific effects. To investigate the molecular mechanisms leading to CAID-related clinical manifestations, we recently performed multiomics studies on primary control versus patient-derived dermal fibroblasts, which pointed to canonical  as well as non-canonical changes in signaling pathways, including disturbed transforming growth-factor beta signaling and altered fibroblast ion-channel function12.SGO1 function is necessary for correct cell division in mammals. In humans, SGO1 somatic mutations are found in colorectal cancers and are associated with chromosomal instabilityf).Here, we show that disordered cardiac pacemaker function in CAID syndrome is attributable to disruption by the SGO1-K23E mutation of an SGO1 non-canonical function to enhance the cardiac pacemaker funny current  monolayers. Lentivirus was used to carry empty vector (control), wild type SGO1 (SGO1-WT) and K23E mutant SGO1 (SGO1-K23E). The numbers of spontaneous beating cultures were counted 30–48 h after lentiviral transduction. We found that SGO1-WT transduced NRVMs had a higher percentage of spontaneously beating cultures compared to control and SGO1-K23E transduced NRVMs Fig. . This ref acts as a pacemaker current in cardiac cells showing spontaneous automaticity, including cells in the principal pacemaker sinoatrial node region, carrying an inward current at negative potentials that initiates spontaneous diastolic depolarization, contributing to the “voltage clock” mechanism of cardiac automaticity13. We therefore considered whether transduction of SGO1 or its K23E mutant affects If in NRVMs. Typical If recordings were obtained from NRVMs, similar to previously reported results14  and L-type (ICaL) calcium currents are involved in diastolic depolarization and the subsequent AP upstroke15. ICaL recording showed no differences among control, SGO1-WT and SGO1-K23E transduced NRVMs . If was compared at day 2–3 after transfection. SGO1 knockdown significantly suppressed If current density  channels . HCN4 contributes up to 70–80% of If in sinoatrial node cells22. We first considered the possibility that, consistent with the canonical role, SGO1 changes HCN channel expression via direct or indirect epigenetic or transcriptional regulation. However, no difference in HCN1, HCN2, HCN3 or HCN4 mRNA expression was observed in response to SGO1-WT and SGO1-K23E transduction of NRVMs  experiments to assess potential interactions between SGO1 and HCN4 in non-transfected native NRVMs. The SGO1-HCN4 complex was pulled down by a rabbit polyclonal antibody against HCN4 and SGO1 bands were detected with a mouse monoclonal antibody against SGO1, which we previously validated to specifically label SGO1 in human and mouse24 . High magnification showed the overlapping staining for SGO1 and HCN4  in CAID-syndrome patients is located in the N-terminal of SGO1 (residues 1-115), which is a highly conserved coiled-coil domain that confers interaction with other cohesin-associated proteins, such as PP2Ae24 Fig. . Two banity Fig. . The cority Fig. . We alsoity Fig. . The SGOity Fig. . These rity Fig. . The whiCN4 Fig. . No staiWT-mCherry) in NRVMs. Rat monoclonal antibodies against mCherry were used to pull down the protein complex and mouse monoclonal antibodies against GFP were used for immunodetection. Immunoprecipitation with mCherry antibodies revealed co-IP of HCN4-GFP and SGO1WT-mCherry in co-expressing NRVMs  and 3 CAID syndrome patients  harboring the homozygous c.69 A > G variant in SGO1  values of 327 and 295 ms respectively, versus normal values of the order of 28–29 ms36.The availability of patient-derived human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CMs) has allowed for powerful in vitro exploration of disease-specific pathophysiological mechanismsMembrane oscillations resembling delayed afterdepolarization (DADs) were observed during longer diastolic intervals for CAID-patient hiPSC-CMs Fig. , arrows.f between control and CAID-patient hiPSC-CMs. The hiPSC-CMs differentiated with our protocol showed robust If  was decreased compared to the healthy control hiPSC-CMs (C1 and C2)  Fig. . No diffC2) Fig. . These rCaL and ICaT in CAID-patient hiPSC-CMs. Typical ICaL recordings were obtained in control and CAID-patient hiPSC-CMs  and SR Ca2+-uptake) take part in cardiac pacemaking49. In this study, we focused on the channels involved in the “membrane clock” mechanism because of clear changes in If. However, we cannot exclude effects mediated by changes in the calcium clock. Further work is needed to evaluate the potential involvement of other factors that might affect pacemaking in CAID patients.Both the “membrane clock”, in which HCN channels play a central role, and the “calcium clock” (involving sarcoplasmic-reticulum (SR) Caf in a direct physical, and cohesin-independent fashion. SGO1 directly interacts with HCN4 and enhances HCN4 cell surface expression, thereby regulating If and pacemaker activity. The K23E mutation in SGO1 results in a loss of interaction between SGO1 and HCN4, impairs HCN4 surface expression, reduces If and disturbs pacemaker activity. These findings shed light on the basis for cardiac pacemaker dysfunction in CAID Syndrome and suggest that improving HCN4 cell surface expression via SGO1 could be a therapeutic approach for pacemaking disorders.In summary, we report a non-canonical role for SGO1, through which SGO1 and SGO1-K23E appear to affect I2 surface area in 24-well plates or 4 mm×10 mm coverslips. In the first 6 h, the cells were cultured in a routine cardiomyocyte medium based on M199 containing 10% FBS and 100 U/ml penicillin/streptomycin, then used for lentivirus transduction in medium with 5% FBS. After 24-h transduction, we refreshed the medium with medium containing 10% FBS and penicillin/streptomycin. After 48-h culture, action potentials were recorded from the NRVM monolayer cultures on coverslips. Single cells were digested from the monolayers with trypsin-EDTA and used for If and ICa recording.All animal-handling procedures followed the National Institutes of Health guidelines and were approved by the Montreal Heart Institute Animals Research Ethics Committee. Neonatal rat ventricular myocytes were obtained from 1 or 2 day old Wistar rat pups (Charles River Laboratories). Neonatal rat pup sex was not determined. Hypothermia was utilized to anesthetize rat pups before they were euthanized by decapitation. The Neonatal Cardiomyocyte Isolation System  was utilized to obtain single cardiomyocytes. To form a monolayer, the NRVMs were plated at a density of 20,000 cells per cmHCN4, human wild type  and mutant (SGO1-K23E) SGO1 were subcloned into the lentiviral vector pLentiCMV in which the GFP coding section was deleted from the backbone of pLentiCMV-GFP-Neo . The GFP was cloned into the N-terminal of HCN4 and mCherry was cloned into the N-terminal of SGO1-WT and SGO1-K23E to form fusion proteins. The lentiviral plasmids carrying SGO1-WT, SGO1-K23E, HCN4, SGO1-WT-mCherry, SGO-K23E-mCherry, or HCN4-GFP were co-transfected into HEK293T cells with packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260), which were gifts from Didier Trono. The supernatants containing virus were harvested 48 and 72 h after transfection and concentrated using Lenti-XTM concentrator (TaKaRa Bio). The viral pellet was resuspended in serum-free DMEM medium and viral titers were measured with QuickTiter Lentivirus Quantitation Kit (Cell Biolabs). Viral stocks were stored at –80 °C until use.The cDNAs of human Lentiviruses were used to overexpress wild type SGO1 and mutant SGO1 (K23E) in NRVMs. The cells were cultured for 6 h to permit cell attachment to the coverslips or plates. For overexpression experiments, the lentiviruses were added to NRVM cultures and incubated for 24 h. The medium was then refreshed with normal medium lacking lentivirus. MOI = 50 was used in overexpression experiments. To knock down SGO1 in NRVMs, the medium was refreshed into Opti-medium without FBS before transfection. The cells were then transfected with siRNA or scrambled RNA at 100 nM per well in 24-well plates with Lipofectamine RNAiMAX (Life Technologies). After 24 h, the Opti-medium was replaced by M199 with 10% FBS. The siRNA  sequences are listed below (5ʹ−3ʹ): 1. UUUCAGUGUACACAGCUGGCAGGUG, 2. CACCUGCCAGCUGUGUACACUGAAA. Stealth RNAi™ siRNA Negative Control Med GC Duplex  was used as scrambled control RNA.50. The cells were superfused with Tyrode Solution containing NaCl 136 mM, KCl 5.4 mM, NaH2PO4 0.33 mM, CaCl2 1.8 mM, MgCl2 1 mM, glucose 10 mM, and Hepes 5 mM . The internal solution contained KCl 120 mM, MgCl2 1 mM, Mg-ATP 3 mM, EGTA 10 mM, Hepes 10 mM . For If recording, the standard whole cell tight seal patch-clamp method was used. To block IK1, ICaL, ICaT and Ito, 2 mM BaCl2, 0.2 mM CdCl2, 2 mM NiCl2 and 4 mM 4-aminopyridine (4-AP) were added to Tyrode solution and with the same internal solution as used for AP recording51. The external solution for ICa recording contained tetraethylammonium chloride (TEA-Cl) 136 mM, CsCl 5.4 mM, CaCl2 2 mM, MgCl2 0.8 mM, Hepes 10 mM, glucose 10 mM and 4-AP 2 mM . The internal solution for ICa recording contained CsCl 120 mM, TEA-Cl 20 mM, MgCl2 1 mM, Mg-ATP 5 mM, Li-GTP 0.1 mM, EGTA 10 and Hepes 10 52. ICaL and ICaT were separated with the use of selective voltage protocols as shown in Fig. Whole cell patch-clamp recordings were performed with an Axopatch 200B amplifier . Data were sampled at 10 Hz and filtered at 5 kHz. All recordings were obtained at 36 °C. Cell capacitance was calculated from the time constant of a capacitative current elicited by a 5 mV depolarization from −60 mV. Borosilicate glass microelectrode tip resistances were 5–8 MΩ. Action potential (AP) recordings were obtained with nystatin perforated patch-clamp pipettes with I = 0 mode as described previously® TGX™ Gels (Bio-Rad Laboratories). Proteins were electrotransferred onto Immobilon-P PVDF membrane . After blocking 1 h in Tris-HCl-buffered saline with 0.2% Tween-20 (TBST) and 5% non-fat milk (Bioshop® Canada), the membranes were incubated overnight with primary antibody in TBST containing 2% non-fat milk. The rabbit polyclonal anti-HCN2 antibody , rabbit polyclonal anti-HCN4 antibody , mouse monoclonal anti-SGO1 antibody , mouse monoclonal anti-GAPDH antibody , mouse monoclonal anti-GFP antibody , rat monoclonal anti-mCherry  and mouse monoclonal anti-PP2A C subunit  were utilized to detect HCN2, HCN4, SGO1, GAPDH, GFP, mCherry and PP2A C proteins respectively. Detection was carried out by the use of horseradish peroxidase conjugated secondary antibodies , photographic film (Agfa NV) and the Western Lighting® plus ECL kit (PerkinElmer).Western blot analysis was performed on Mini-PROTEAN® RNA Kits (Macherey-Nagel). First-strand complementary DNA was synthesized from 2 μg of total RNA with high-capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time qPCR was carried out with carboxy-fluorescein (FAM)-labeled fluorogenic TaqMan assay primers and TaqMan Universal Master Mix (Applied Biosystems). Fluorescence signals were detected with StepOnePlus™ Real-Time PCR System (Applied Biosystems) in duplicate and relative quantities (2-ΔCt) were calculated with the geometric mean of the reference gene GAPDH as an internal standard. TaqMan® Assay primers (ThermoFisher) used for testing HCN1, HCN2, HCN3, HCN4 and GAPDH are list in Supplementary Table RNA was isolated with NucleoSpinCells were plated on 0.2% gelatin-coated coverslips and cultured for 3 days. Cultured cells were washed twice with 0.1 M phosphate-buffered saline (PBS) and fixed with paraformaldehyde (4%) in PBS for 10 min at room temperature, followed by several washes in PBS. Cells were subsequently incubated in PBS containing 2% normal goat serum for 30 min to block non-specific binding. They were then incubated with rabbit polyclonal anti-HCN4 antibody  mixed with mouse monoclonal anti-SGO1  in 2% normal goat serum PBS at 4 °C overnight. After washing with PBS, the cells were incubated with Alexa 488-conjugated goat anti-rabbit secondary antibody  and an Alexa 555-conjugated goat anti-mouse secondary antibody  for 1 h at room temperature. Nuclei were stained with 1 µM TOPRO3. Negative controls were performed by omitting the primary antibodies from the incubation solution. The fluorescence images were obtained with a Zeiss LSM‐710 inverted confocal laser scanning microscope  and ZEN2.6 software was utilized for image collection.3VO4. The cells were treated homogenized with an ultrasonic device. The homogenates were centrifuged at 12,000 g for 20 min at 4 °C. Supernatants were collected and protein concentration was determined by the Bio-Rad Protein Assay Dye Kit . Cell homogenates (400 μg of protein) were preincubated for 1 h with 20 μl of protein G-Sepharose CL-4B (Amersham Biosciences) at 4 °C and centrifuged to remove proteins that had adhered nonspecifically to protein G. In total, 4 μg primary antibodies bound to and then were cross-linked to Dynabeads Protein G  with 20 mM dimethyl pimelimidate (DMP). The supernatants were incubated with the Dynabeads-protein G-antibody complex overnight at 4 °C. The supernatant was removed by magnet and the pellets were washed three times with co-IP buffer without glycerol. Bound proteins were eluted by boiling at 60 °C for 9 min in 2×SDS-PAGE loading buffer and then isolated by magnet. The proteins in supernatants were assayed by Western blot. Rabbit polyclonal anti-HCN4 antibody  and mouse monoclonal anti-SGO1 antibody  were utilized to pull down the protein complex. As an isotype control, mouse monoclonal anti-phospholaman (PLN) antibody , mouse monoclonal anti glycogen synthase 1 antibody  and normal rabbit IgG  were used in co-IP experiments.NRVMs or hiPSC-CMs were harvested in ice-cold co-IP buffer containing 50 mM Hepes buffer (pH 7.4), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, protease inhibitor cocktail , 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na2+ and Ca2+ at pH 7.3. Membrane proteins were biotinylated by incubating cells with 2 mg/mL of EZ-linkTM sulfo-NHS-LCLC-biotin (Thermo Fisher Scientific) in DPBS for 30 minutes at 4 °C with occasional shaking. The cells were then rapidly washed with DPBS 3 times, then washed with DPBS containing 50 mM glycine to allow neutralization of non-crosslinked biotin reagents. The cells were then scraped into IP buffer and rotated for 1 h at 4 °C. The insoluble materials were removed by centrifugation . The supernatants were mixed with Ultralink Immobilised NeutrAvidin beads (Thermo Fisher Scientific) and rotated overnight at 4 °C. The co-IP buffer without glycerol was used to wash the beads for 45 min. Precipitated beads were resuspended in 2×SDS-PAGE loading buffer and heated for 9 min at 60 °C. Subsequently, Western blots were performed to analyze biotinylated HCN4.NRVMs were washed with Dulbecco’s Phosphate-Buffered Saline (DPBS) containing MgTM Flex medium on Matrigel® (Corning) at 5% CO2 37 °C incubator. The pluripotency was tested with RT-qPCR for marker genes using FastStart Universal SYBR Green (Roche). The primer sequences are listed in Supplementary Table Human induced pluripotent stem cells (hiPSC) were derived from two healthy donors: C1 (SJi3252C2) and C2 (SJ3013C2) and three CAID patients homozygous for SGO1-K23E: M2 (SJi2551C2), M4 (SJi2690C2), and M5 (SJi2391C2). All participants gave written informed consent. The use of pluripotent stem cells was approved by the Research Ethics Committee of Sainte Justine University Hospital Center and the Ethics Committee of Montreal Heart Institute and carried out in accordance with guidelines from the Fonds de Recherche en Santé de Québec and the Declaration of Helsinki. The hiPSCs were generated from dermal fibroblasts at CHU Sainte Justine Stem Cell core using CytotuneTM 1.0 or 2.0 (Invitrogen) following manufacturer recommendation. The cell lines were then maintained in Essential 853. Briefly, hiPSC colonies were passaged with 0.5 mM EDTA-PBS solution and seeded onto Matrigel-coated plates in E8 medium containing 5 µM Y27632 ROCK inhibitor (Selleckchem). The cells reached 70% cell confluence after 3 days in culture and were then treated with 5 µM CHIR99021 (Selleckchem) in RPMI1640 (Gibco) + B27 supplement for 1 day. On day 2, the cells were treated with 5 µM CHIR99021 + 10 ng recombinant human BMP-2 (Peprotech) + 1 µM SU5402 (Selleckchem) in RPMI1640 + B27 supplement. On day 3, the cells were treated with 10 µM IWP2 (Selleckchem) + 10 ng recombinant human BMP-2 + 1 µM SU5402 in RPMI1640 + B27 supplement. From day 4 to day 5 the cells were placed on the RPMI1640 + B27 supplement with 10 µM IWP2. On days 6 to11, the IWP2 was removed from the medium and the cells were cultured in Ham’s F12 medium + 5% KnockOut™ Serum Replacement (Thermo Fisher Scientific) with 2 ng FGF2 (Recombinant Human FGF-basic (146 a.a.), Peprotech). From day 12 onward, the cells were cultured in medium M199 + 5% Serum Replacement with 2 ng FGF2 and the media were refreshed every 2 days. To further purify the hiPSC-CMs for the Western blot and co-IP experiments, at day 21 the cells were starved for 3 days in glucose-free RPMI1640 + 5% Serum Replacement with 2 ng FGF2. Then the cells were placed on M199 + Serum Replacement with 2 ng FGF2 for 2 days and collected. For the patch-clamp experiments, the hiPSC-CMs were dissociated with TrypLE™ Select Enzyme  into single-cells, resuspended and replated on coverslips coated by Matrigel. After 2 days for recovery, the cells were utilized in patch-clamp experiments.A modified highly efficient differentiation method was used to produce beating monolayers2, absorbance was measured using BioTek ELx800 Absorbance Microplate Reader (BioTek Instruments) at 570 nm and normalized to 600 nm values (reference wavelength). Absorbance readings were used to calculate for the cell viability for each group following manufacturer recommendation.PrestoBlue™  resazurin-based assay was utilized to determine NRVMs viability after the SGO1 knockdown 2 days. In total, 10 μL PrestoBlue reagent (/well) was added to 90 μL media for one well (96-well plate); the wells containing only media were used to correct for background color. After 30 min of incubation at 37 °C, 5% COt test was performed for comparison of 2 groups only. P < 0.05 was considered statistically significant; 2-tailed tests were used for all analyses. Analysis of the Western blot bands was performed with ImageJ 1.52 P image analysis software . All measurements were obtained from distinct samples. Patch-clamp data were analyzed with Clampfit 9.0 . GraphPad Prism 3.0  was used for other data analyses.Data are reported as the mean ± SEM. Multiple group statistical comparisons for I-V relations were obtained by 2-way ANOVA. Statistical analysis of the results of >2 groups was carried out by one-way ANOVA followed by the least significant difference test or Tukey test. Student’s Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary