Breast cancer screening has substantially reduced mortality across screening populations. However, a clinical need persists for more accessible, cost-effective, and robust approaches for increased-risk and diverse patient populations, especially those with dense breasts where screening mammography is suboptimal. We developed and validated a cost-effective, portable, patient-dedicated three-dimensional (3D) automated breast ultrasound (ABUS) system for point-of-care breast cancer screening. The 3D ABUS system contains a wearable, rapid-prototype 3D-printed dam assembly, a compression assembly, and a computer-driven 3DUS scanner, adaptable to any commercially available US machine and transducer. Acquisition is operator-agnostic, involves a 40-second scan time, and provides multiplanar 3D visualization for whole-breast assessment. Geometric reconstruction accuracy was evaluated with a 3D grid phantom and tissue-mimicking breast phantoms, demonstrating linear measurement and volumetric reconstruction errors < 0.2 mm and < 3%, respectively. The system’s capability was demonstrated in a healthy male volunteer and two healthy female volunteers, representing diverse patient geometries and breast sizes. The system enables comfortable ultrasonic coupling and tissue stabilization, with adjustable compression to improve image quality while alleviating discomfort. Moreover, the system effectively mitigates breathing and motion, since its assembly affixes directly onto the patient. While future studies are still required to evaluate the impact on current clinical practices and workflow, the 3D ABUS system shows potential for adoption as an alternative, cost-effective, dedicated point-of-care breast cancer screening approach for increased-risk populations and limited-resource settings. While breast cancer has a high incidence, it has been shown to have a relatively favorable survival rate, which can be attributed to the detection of early-stage breast cancers through screening in predominantly developed countries4. Mammography is undoubtedly an important factor in reducing breast cancer-related mortality, notably, in screening populations over 50 years of age and those assigned female at birth5. However, population-based screening protocols and recommendation guidelines differ widely across population age, frequency, and modalities6. Beyond screening average-risk populations, there exist several other groups and risk-factors that increase the overall risk for developing breast cancer. These specific risks broadly include hereditary breast cancer, with inherited BRCA1 or BRCA2 gene mutations, a personal or family history of breast cancer, a history of chest radiation, and dense breasts8. Moreover, younger women are more likely to develop more aggressive breast cancers, which often present at more advanced stages at the time of detection and diagnosis11. Across these intermediate to high-risk populations, current breast cancer screening recommendations and protocols lack complete consensus and may be suboptimal12.Globally, breast cancer is the most commonly diagnosed cancer in women, with over 2.3 million women newly diagnosed and 650,000 deaths in 20208. This limitation may potentially result in delayed diagnosis between recommended screens and interval cancers, and poorer overall prognosis in these women with dense breasts13. Moreover, breast density alone is a strong, independent risk-factor for the development of breast cancer13. While in North America, the proportion of women with dense breasts represents approximately 40% of all screening population14, the proportion is substantially higher amongst diverse ethnic and racial populations16. Due to the aforementioned challenges in screening increased-risk women with dense breasts, supplemental screening with magnetic resonance imaging (MRI), digital breast tomosynthesis (DBT), and ultrasound (US) have been effectively used to improve early detection of breast cancer18. Despite its clinical utility and potential diagnostic advantages, the value of supplemental screening with these modalities remains unclear7. Furthermore, the required facilities, specialized healthcare operators and infrastructure needs, associated costs, and contraindications limit their widespread use as accessible screening modalities, especially in limited-resource settings.In women with dense breasts, which refers to an increased proportion of fibroglandular tissues compared with fatty adipose tissues, the mammographic sensitivity to detect breast cancer is substantially reduced. Increased breast density has a masking effect in mammograms, due to challenges in discriminating between dense breast tissues and malignancies19, and preventative health behavior, including breast cancer screening, is substantially lower due to barriers to care20. Specifically, there exists an elevated risk for developing breast cancer in these minority populations, compared with their heterosexual counterparts21. These individuals and groups are less likely to participate in recommended screening, as they often lack equitable and inclusive practices, which may ultimately result in diagnosis of breast cancer at more advanced stages, where intervention and treatments are less successful22.Beyond established risk-factors, there are clear disparities in breast cancer screening in several equity-seeking and marginalized populations, resulting in a disproportionate increase in cancer-related morbidities. While the term ‘women’ has been widely used in clinical practice and literature, as those who are assigned female at birth, other sexual and gender minorities, such as transgender or nonbinary individuals, may have similar associated and increased risk-factors as cisgender women. In sexual and gender minorities, evidence-based recommended cancer screening guidelines are unclearCancer-related health inequities stem from structural marginalization in race and ethnicity, sexual orientation and gender, and other social determinants of health, including socioeconomic status and environmental factors  that limit accessibility, impose cost constraints and reduce inclusivity of these individuals and groups in cancer care. For many of these populations, point-of-care technologies can provide a practical solution by allowing for data acquisition at the site of care and actionable information for rapid clinical decision-making, alleviating challenges associated with limited-resource or cost-constrained healthcare settings. Therefore, the development of more accessible, cost-effective, and point-of-care technologies for early-detection and diagnosis in increased-risk populations has the potential to advance breast cancer-related health equities and outcomes.23. With increased-risk populations, specifically those with dense breasts, handheld (HH) US has been shown to be an effective supplemental screening method for detecting small, early-stage, and invasive breast cancers26. However, HHUS is operator-dependent and time-consuming, which limit its use as a widespread screening modality. Automated breast (AB) US is an emerging method that shows utility for supplemental screening in women with dense breasts27. ABUS imaging allows for more standardized acquisition, improved operator-dependence, and reproducibility with three-dimensional (3D) volumetric whole-breast assessment29. Most common, commercially available ABUS systems employed in clinical practice are the Invenia ABUS  and ACUSON S2000 Automated Breast Volume Scanner (ABVS) . These ABUS systems involve a supine acquisition approach and are comprised of an external articulated manipulator, ultrawide high-frequency 5–14 MHz US transducer, touchscreen monitor (scan station) and workstation (view station) for 3D image reconstruction, visualization, and interpretation. The ABUS image acquisition involves a computer-driven mechanical scan to automatically translate the ultrawide US transducer within a sonolucent compression paddle, with a disposable membrane for coupling. Acquisition typically involves three views: anteroposterior (AP), lateral (LAT), and medial (MED) views in each breast, with additional image views acquired to expand the volumetric coverage in larger breasts, which takes approximately a 10-min scan time27.Ultrasound (US) is a widely available, non-ionizing, compact, and cost-effective imaging modality in clinical practice30, there are several limitations. High quality ABUS requires operator training for proper patient positioning and ultrasonic coupling, in order to avoid artifacts during image acquisition32. Moreover, the US transducer paddle is not designed to fit all women and their breasts, nor is adaptable to different patient populations30. The ABUS acquisition has also been reported as painful by some patients due to the required compression from ultrawide US transducers, which are required to obtain reliable images33. Although these limitations can be mitigated with increased operator training for patient positioning and careful acquisition protocols, alternative, more robust approaches are still necessary32. Beyond these technical limitations, most commercially available ABUS systems are relatively bulky and costly, as they require complete machines with articulated manipulators, costly ultrawide US transducers, or specialized patient examination tables for prone acquisition approaches34.While currently available 3D ABUS approaches have shown clear clinical utility and high diagnostic performance for supplemental screening, especially in women with dense breastsTo address these aforementioned challenges, this work aims to develop an alternative, cost-effective, portable, and patient-dedicated 3D ABUS system for point-of-care breast cancer screening with utility in increased-risk populations and limited-resource settings. The proposed 3D ABUS system utilizes a rapid-prototype, dam assembly to conform to the patient’s breast and contain coupling gel for a supine acquisition approach. This paper describes the design and fabrication of the dedicated 3D ABUS system, reports on experimental validation in various test phantoms, and demonstrates the system’s utility in three proof-of-concept healthy volunteers, representing diverse patient geometries and breast sizes. Importantly, the 3D ABUS system can accommodate any commercially available US transducer, utilizes rapid-prototyping, 3D-printing, and economical materials, opening the door to personalized, patient-specific 3D ABUS imaging. The 3D ABUS acquisition approach removes operator-dependence to acquire high-resolution 3DUS images. Moreover, it is capable of point-of-care imaging with real-time multiplanar 3D visualization enabling online or offline whole-breast assessment. Lastly, we demonstrated the 3D ABUS system’s versatility and robustness across various parameters and acquisition conditions, improving ultrasonic coupling, tissue stabilization for image acquisition, and mitigating artifacts due to patient breathing and motion.35.The 3D ABUS system was fabricated entirely with rapid-prototyping, 3D-printing, and low-cost economical materials, including Delrin, Aluminum, and Brass, commercially available linear bearings and electronic components. The 3D-printed components were fabricated with a 3D Idea Builder 3D40 3D Printer  and 1.75 mm diameter PLA filament, which were printed with a standard resolution of 0.20 mm. The adaptable US transducer cradle can accommodate any commercially available 2D linear array US transducer from any US machine. The approximate weight of each constituent component is 613 g for the wearable dam assembly, 397 g for the compression assembly, and 500 g for the motorized scanner. The total weight of the prototype 3D ABUS system is 1510 g without the linear US transducer, and approximately 1625 g with an integrated Canon  14L5 (PLT-1005BT) (10 MHz) linear array transducer.The dedicated 3D ABUS system, shown in Fig. The dedicated 3D ABUS system is a portable, compact system that is entirely contained on the wearable dam assembly, which serves as the base of the system and is affixed directly onto the patient with adjustable nylon webbings and quick-release buckles. The system’s dam assembly is positioned around breast, with the opening for the scanning assembly accessible from the anterior side of the patient, allowing for supine positioning for image acquisition. The wearable dam is a personalizable 3D-printed component that is shaped like a hollow square with rounded corners, Fig. c with a The curvilinear base, as shown in Fig. The dam assembly further supports an optional adjustable compression plate assembly and the motorized 3DUS scanner. The compression plate assembly is mounted on the top surface of the dam assembly and secured with a clamping screw. The compression plate is made from a 10.0 × 10.0 cm TPX polymethylpentene (PMP)  and is supported with a thin aluminum frame, which is positioned above the breast and parallel to the chest wall. The assembly contains an adjustable turn knob mechanism to manually elevate or lower the TPX plate for immobilization or compression of the breast. A light compression would stabilize the breast and eliminate any air gaps or bubbles in the US transmission gel. An increased compression would reduce tissue thickness, without painful application or discomfort, to homogenously distribute the breast tissues and improve image quality by allowing increased US frequency by reducing the imaging depth.The 3D ABUS scanner consists of a computer-driven motorized-drive mechanism  on a stainless-steel linear stage  for hands-free, operator-independent, automated 3DUS image acquisition. The linear motor stage employed in our prototype 3D ABUS system is affixed to the patient dam with a positioning accuracy of ± 0.06 mm and allows a maximum travel distance of 104.5 mm for the scan head to translate along the rail in one direction, as shown in Fig. 36. Therefore, the 3D spatial resolution of the resultant 3DUS image is a combination of the native in-plane  resolution of the acquired 2DUS images, and out-of-plane  resolution, as determined by the native elevational resolution of the US transducer and frame spatial density of the acquired 2DUS images. If the spatial density is sufficiently high, as determined experimentally, the out-of-plane resolution is equivalent to the native elevational resolution of the US transducer.Three-dimensional ABUS image acquisition involves activating the motorized linear scanner to automatically translate the US transducer perpendicular to the 2DUS image plane  to a specified scan distance, depending on the specific 3D-printed dam assembly window opening and US transducer dimension constraints. During 3DUS image acquisition, spatially encoded 2DUS images are continually acquired at a fixed and adjustable spatial interval  as the US transducer is translated along its track. The 2DUS images are acquired into any PC via an Epiphan Digital Visual Interface (DVI) to USB interface to video frame grabber . As the 2DUS images are collected, a 3DUS image is reconstructed in real-time, providing a 3D ABUS image at the completion of the scan periodThe removable motorized 3D ABUS scanner can be used in one of two orthogonal configurations: (a) craniocaudal direction to acquire transverse 2DUS images, or (b) mediolateral direction to acquire sagittal 2DUS images Fig. d. The 3D37. This VBA determines the intensity value of a fused voxel based on the relative distance to an edge in the overlapped 3D volume. Since the position and orientation of the 3DUS image volumes are known, the weighting factors can be pre-determined to optimize computational speed when fusing multiple 3DUS images and assigning overlapping voxel values. The final combined 3D ABUS image depicts a whole-breast volume that can be viewed and manipulated in any direction and orientation using our in-house 3D multiplanar viewing software.While the scan distance, in the elevational direction of the US beam, can be adjusted to acquire an increased volumetric region, since the volumetric field-of-view of most conventional linear US transducers are unable to cover the entire breast during a single 3DUS scan, the US cradle, containing the US transducer, can be shifted laterally  on the mounting rail to another preset position. Once the transducer has been relocated relative to the first 3DUS scan, the motorized scanner is activated again to acquire a second 3DUS image. Since the two 3DUS images are at a known position (distance and orientation) relative to each other, this allowed the software to combine the acquired 3DUS images with a transformation matrix. The combined whole-breast 3DUS image was reconstructed by transforming each 3D voxel position into the image coordinate space, which corresponds to the tracked position and orientation of the acquired images. Two or more parallel and partially overlapping 3DUS images then can be registered and fused in real-time using a rigid registration and weighted voxel-based algorithm (VBA) that assigns the intensity values of overlapping 3D voxelsGeometric reconstruction accuracy of the acquired 3D ABUS images was verified with both linear and volumetric measurements. For these experiments, the compression assembly and sonolucent membrane were not included on the 3D ABUS system.38. The phantom was immersed in a solution made from a mixture of distilled water and 7.25% by volume isopropyl alcohol to mimic the approximate speed-of-sound in soft tissues of 1540 m s−139. The 3D ABUS images were acquired at 25 frames s−1, 6 frames mm−1, 80 mm scan distance, 40 mm depth setting, and 29.5 mm lateral translation, resulting in a 3DUS image volume of 80 × 87.5 × 40 mm3 and voxel size of 0.14 × 0.14 × 0.33 mm3.Linear reconstruction and measurement accuracy in each US image dimension was validated with a custom 3D grid phantom. The 3D grid phantom is made from crosshatched 0.1 mm diameter monofilament polyester wires with a 10.0 × 10.0 mm grid, positioned in four parallel layers spaced 10.0 mm apart in the elevational direction, as shown in Fig. Linear accuracy measurements were obtained using in-house software workstation for visualization of the 3DUS images. To acquire linear measurements, the 3DUS image was visualized in the axial or sagittal view plane of interest. Linear reconstruction errors were computed by performing distance measurements N = 22, each) between the center of two adjacent monofilament wires in the lateral, elevational, and axial  US directions, illustrated in Fig. 2, each bThe limits of the in-plane resolution  of our 3D ABUS system were determined using a CIRS  model ATS539 phantom. Similarly, using a CIRS model 538NH phantom, the elevational resolution limits were examined.3 in volume) was fabricated with a mixture of 3% agar powder per mass, 8% glycerol per mass, and distilled water to mimic the speed-of-sound in soft tissues of approximately 1540 ms−139. To simulate parenchymal breast tissues, 15% per mass SigmaCell cellulose scattering agent  was added to the background mixture. Agar-based small and medium spheres (4.76 and 9.53 mm in diameter) were fabricated with a 3D-printed template without added scattering agent and embedded into the breast phantom to appear hypoechoic relative to the background ‘tissue’ for identification and volumetric measurements.An agar-based tissue-mimicking breast phantom  from its actual known volumes were computed with Eq.  in the Segment Editor moduleThe utility of the dedicated 3D ABUS system was evaluated in three healthy volunteers with written informed consent and the study was approved by the Western University  Health Sciences Research Ethics Board  at the Schulich School of Medicine and Dentistry. All healthy volunteer studies and methods were performed in accordance with the relevant guidelines and regulations of the HSREB. A depiction of the dedicated 3D ABUS system positioned on a healthy female volunteer is shown in Fig. To evaluate our 3D ABUS approach across a broad range of diverse patient geometries and breast sizes, we imaged one healthy male volunteer  and two healthy female volunteers, one with a 34A breast cup size  and the second with a 32F breast cup size . The healthy volunteers also provided informal feedback to evaluate the 3D ABUS system, specifically regarding patient comfort and functionality. Comfort evaluation included the physical ease of wearing the dam assembly, including the shape of the curvilinear dam base and the flexible tubing material. Functionality assessment included evaluating the effectiveness of the system in conforming to the natural curvature and shape of various breast sizes and geometries, allowing for the stabilization of the breast during 3D ABUS image acquisition.A replaceable sonolucent membrane was attached onto the wearable dam assembly, then a US lotion was applied on the skin-facing surface for coupling.The wearable dam assembly, as the base of the dedicated 3D ABUS system, was affixed to the patient over either the left or right breast in a comfortable, sitting or standing position. The nylon webbing straps were fastened around the patient, such that the dam assembly was positioned securely on the breast.The patient lay supine, then low viscosity US transmission gel was poured into the dam assembly to cover the interior volume of the dam over the breast.The optional compression assembly was positioned on the dam assembly. The knob was manually adjusted to lower the TPX compression plate to immobilize the breast with a desired compression level, without painful application.The motorized 3D ABUS scanner was rigidly mounted on the top plate with the locating pins and stabilization magnets in either the (a) craniocaudal direction to acquire transverse 2DUS images or (b) mediolateral direction to acquire sagittal 2DUS images. This setup process from Step 1 to 5 takes approximately 10-min.The US acquisition parameters, including the scan distance, lateral translation, depth setting, frame density, and frame rates were adjusted by a trained operator.Computer-driven 3D ABUS image acquisition was performed by activating the motorized linear scanner with a simple button in the software workstation. The US cradle was translated laterally to a second preset position(s) to expand the volumetric field-of-view for whole-breast coverage. The scanning time for a single pass is up to 20-s, depending on the acquisition parameters, with a total maximum acquisition time of 40-s. The repositioning of the US cradle to another position is immediate and takes approximately one second.The parallel and partially overlapping 3DUS image(s) were registered in real-time for bedside or offline whole-breast assessment with multiplanar 3D views in axial, sagittal, and coronal view planes, or oblique planes.The cleanup involves the removal of the motorized scanner and dam assembly, entirely containing the ultrasonic coupling gel, and wiping off any remaining sonolucent lotion from the breast. This process takes approximately one-minute.The proposed clinical workflow to perform 3D ABUS imaging in a healthy volunteers or patients, including preparation, patient positioning, image acquisition, and cleanup was as follows:−1, 4 frames mm−1, 80.0–90.0 mm scan distance, and 40.0–60.0 mm depth setting, resulting in a 3DUS image volume of 80.0–90.0 × 87.5 × 40.0–60.0 mm3 and voxel size of 0.14 × 0.14 × 0.33 mm3. The total time to acquire a 3D ABUS image with these parameters was approximately 20–28 s: 10–14 s per each pass. In addition to acquiring dedicated 3D ABUS images of the healthy volunteers, the following parameters and acquisition variables were evaluated for their impact on 3D ABUS image quality: (a) use of the sonolucent membrane, (b) use of the compression assembly, and (c) effect of patient breathing and motion.A wearable 3D-printed dam assembly with an approximately 1-inch wall at its maximum curvature height was used for the healthy male volunteer and healthy female volunteer with the 34A breast size. A second 3D-printed dam assembly with a 3-inch wall was used for the second healthy female volunteer with a 32F breast size. Feedback from the volunteers highlighted aspects of the 3D ABUS system that required adjustment for enhancing comfortability and functionality, as discussed in later sections of this work. After modifications were implemented, the volunteers all self-reported that the dam fit comfortably against their chest wall, with no leakage of US gel outside of the dam assembly. All 3D ABUS images were acquired at 25 frames sThe 3D ABUS images were acquired with and without the use of the sonolucent membrane, as described in Step (1), to evaluate the impact on image quality. With the sonolucent membrane, US lotion was used to couple the membrane with the breast prior to affixing the 3D ABUS device. Standard low-viscosity US transmission gel was used to fill the volume inside the dam assembly in both cases. The optional compression assembly was not included in either case.3D ABUS images were acquired with and without the use of the optional compression assembly, as described in Step (4). With the compression assembly, minimal compression was used to immobilize the breast (without painful application) to distribute the breast tissues across the acquired volumetric field-of-view. The 3D ABUS image quality was evaluated by a trained observer, and patient comfort or discomfort was qualitatively reported by the healthy female volunteer.The effect of patient breathing was assessed by acquiring 3D ABUS images with (a) breath-hold during 3D ABUS image acquisition and (b) deep-breathing from maximum inspiration to maximum expiration during 3D ABUS acquisition. Both experiments were performed without the compression assembly, as described in Step (4). The effect of patient motion was further assessed by instructing the volunteer to actively talk during the 3D ABUS image acquisition, representing a high level of motion in clinical practice.The mean and SD linear distance measurement errors, and 95% confidence intervals compared with the nominal measurements 10.0 mm) for lateral, elevational, and axial US directions  are summarized in Table 0.0 mm fo3 and 9.44 ± 7.84 mm3, which corresponded to a percent difference of 2.23% and 2.11%, respectively. The overall volumetric percent error across all spherical inclusions in the breast phantom was 2.18%.The volumetric reconstruction and measurements of the small and medium sized spherical inclusions (4.76 mm and 9.53 mm in diameter) in the tissue-mimicking breast phantom are summarized in Table The reports from our healthy volunteer focus group initiated some changes in our system design, specifically the wearable dam assembly. First, we adjusted the curvilinear shape of the patient dam to enhance its conformity to the natural curvature of the chest wall, while accommodating various breast sizes and shapes. Volunteers indicated that the medial side of the dam, which runs parallel to and positioned on the sternum, should be slightly shallower compared to the lateral side. This adjustment is intended to account for potential variations in the chest wall’s natural shape and to optimize the fit of the dam for both breasts. Additionally, the flexible tubing on the base of the dam assembly should be made from a softer material for improved conformity across different body types, allowing for more flexibility between different shapes and variations between breasts. In response to this feedback, the dam assembly underwent minor refinements to modify the curvilinear base dimensions and to include a softer neoprene tubing material. In addition, volunteer feedback provided insights on the adjustability of the dam assembly, specifically pertaining to the straps and fastening mechanism. Alterations were made to include adjustable mechanisms on the strap webbing and the fastening mechanism, allowing for quick, secure, and modifiable attachment of the dam assembly to the volunteer. Feedback from volunteers after the dam assembly alterations indicated that the overall user experience was improved, and initial discomfort of the wearable device was alleviated. This optimized dam assembly was utilized in all three healthy volunteers, with adjustments to the maximum height to encompass breast size.41. These anatomical features and details are clearly visualized as distinct layers in the 3DUS images based on characteristic differences in sonographic appearance from hypoechoic  to hyperechoic  structures42. All volunteer 3D ABUS images show the breast in a natural, supine anatomical position since there is no compression or transducer pressure required when acquiring the images, unlike with conventional HHUS or commercial ABUS applications.The 3D ABUS images acquired in the healthy male and healthy female 34A and 32F) volunteers are shown in Figs. A and 32F3D ABUS images acquired with and without the use of the sonolucent membrane are shown in Fig. Figure 3D ABUS images were further acquired in a healthy female volunteer with a breath-hold and while deep-breathing from a complete inspiration to expiration during acquisition, as an extreme case of breathing and motion, shown in Fig. 28. Beyond these advantages, there are several key design features of our proposed 3D ABUS system that overcome some limitations of alternative ABUS systems. Commercially available systems are typically costly and bulky, as they require a complete dedicated system and workstation, including articulated manipulators, specialized US transducers32, or specialized patient examination tables for prone acquisition34, which limit its overall ease of portability. An alternative, emerging approach that addresses some of these challenges is the ATUSA system , which allows for portable ABUS image acquisition with a compact wearable accessory with an integrated specialized US scanning mechanism43. However, our dedicated 3D ABUS system is cost-effective, adaptable, and versatile. Specifically, our system is fabricated from entirely economical materials and utilizes rapid-prototyping and 3D-printing to accommodate any commercially available US machine and transducer. This maximizes the ease of integration of the dedicated 3D ABUS system into most existing clinical settings, which includes limited-resource or cost-constrained settings, as US imaging is a widely available and accessible modality in clinical practice. When considering settings without availability to US machines, the 3D ABUS system can be adapted to accommodate transducers with portable US machines, which would further improve its transportability and point-of-care applicability.We developed, validated, and investigated the potential utility of a dedicated 3D ABUS system. Our 3D ABUS system exemplifies the advantages of ABUS systems, enabling standardized acquisition and potentially reduced operator-dependence and increased time efficiency, and volumetric 3D visualization for whole-breast assessmentIn addition, the dedicated 3D ABUS system is a portable unit that removes the need for external components, unlike most commercially available ABUS systems. Moreover, its constituent components  are compact and its assembly is relatively intuitive, since all components can be easily attached or removed from the wearable dam assembly that conforms to the patient and serves as the base. Since the 3D ABUS acquisition approach utilizes a supine positioning approach, the natural shape and curvature of the breast is maintained, potentially allowing for correlation with alternative imaging modalities, such as CT or MRI, when similarly acquired in a supine position. Furthermore, the 3D ABUS system is a patient-specific device, as its wearable base assembly can be personalized to conform to the patient or specific patient populations. Specifically, its curvilinear base can be adapted with rapid-prototyping and the flexible, skin-safe Neoprene tube on the underside of the dam  to accommodate diverse populations, including variable patient geometries, and sizes. This design consideration potentially opens doors for more inclusive practices to advance gender-affirming care in sexual and gender diverse populations. Importantly, another advantage of our proposed 3D ABUS design is the ability to acquire and visualize whole-breast images within a single 3D volume. In comparison with alternative, existing commercially available systems, multiple scans at various angles and geometries are required to encompass the entity of the breast. Our proposed system, instead, places the entire breast in a natural position within the dam assembly, improving image coverage during acquisition. The portability, cost-effectiveness, and patient-specific features of the 3D ABUS system allows for utility across various settings, including limited-resource or underserved populations, where advanced technologies or techniques may not be easily accessible.45. Since the volumetric measurements were blinded to the observer prior to comparison with their known volumes, potential biases were minimized, increasing the reliability and validity of this finding. Geometric reconstruction accuracy is necessary to enable reliable quantitative breast lesion measurements and volumetric estimation in clinical practice, which are important diagnostic and prognostic factors in breast cancer management46. Moreover, accurate 3D measurements in axial, sagittal, and coronal view planes may allow for increased accuracy in clinical applications, overcoming limitations associated with operator-dependent HHUS46.In addition to the realization and fabrication of the 3D ABUS system, we experimentally validated its geometric reconstruction accuracy with various phantoms. With a 3D grid phantom, the linear measurement errors showed excellent accuracy and reliability. While there were no substantial differences between the linear measurement directions in the 3D ABUS image, the maximum error in the axial (z-axis) direction may be attributed to the increased difficulty to discriminate the central axis of the wires due to the deterioration of axial resolution from the focal zone of the US beam. Additionally, there were small reverberation artifacts in the monofilament wires, which may have accounted for some uncertainty in the linear measurement errors. While the linear measurements demonstrated excellent accuracy and reliability, it should be noted that these measurements were susceptible to potential bias, since the observer performed linear measurements with a software module that displayed the distances in real-time. This may introduce perception biases and overestimate the accuracy and precision of the linear distance measurements. 3D ABUS imaging was also demonstrated in a tissue-mimicking breast phantom. The small volumetric reconstruction error of the spherical inclusions in the acquired 3D ABUS images are consistent with previously validated mechanically-driven 3DUS acquisition approachesThe 3D ABUS system and its proposed clinical workflow was demonstrated in three healthy volunteers, as a proof-of-concept toward bench-to-bedside translation for breast cancer screening and diagnostic applications. The three recruited healthy volunteers represented a broad range of diverse patient geometries, shapes, and sizes. Feedback from the healthy volunteer focus group indicated critical system requirements for improved user experience and reduced discomfort, which would translate to critical patient requirements when implemented into clinical practice. Additional requirements that will be evaluated in a future study include whole-breast imaging coverage compared with gold-standard modalities, ease of use, and other clinical workflow considerations. Importantly, iterative modifications that were made to the system design to improve patient comfort and functionality in this study further inform the utility of the 3D ABUS system as a wearable design, compared with external imaging systems that may use painful compression techniques during image acquisition. Regarding the symmetry of the dam assembly for 3D ABUS acquisition, the current dam assembly can adequately be positioned on both breasts. However, to address the potential barrier of bilateral asymmetry, particularly with prospective 3D ABUS system designs involving a dam assembly on each breast to streamline workflow, further design refinements may be warranted to optimize the fit on both breasts. While the healthy volunteer feedback provided valuable insights for image acquisition, future studies are necessary to evaluate our prototype 3D ABUS system in a relevant clinical context.41. The healthy female volunteers demonstrated a wide range of breast sizes (34A and 32F), representing substantial differences in breast tissue projection or expansion (bust measurement) from the chest wall (band measurement) of 1-inch and 6-inches, respectively. This demonstrated the capability of the proposed 3D ABUS approach to acquire images in both smaller and larger breasts (A–F breast cup sizes) with this proof-of-concept, with minimal modifications to the personalizable 3D-printed dam assembly. Across all three healthy volunteers, the proposed 3D ABUS imaging workflow  could further mitigate breathing and motion artifacts by moving synchronously with the patient as they breathe and move. Importantly, optimizing 3D ABUS image quality with free breathing would remove the dependence for complex breath-hold methods, further improving overall patient comfort and simplifying workflow in clinical practice.All 3D ABUS images of the healthy volunteers Fig.  were acq51. However, in some cases, there was substantial nipple shadowing and attenuation in the central region of the breast, which resulted in an anechoic region in the 3D ABUS image, as shown in Fig. 52. Importantly, the compression assembly was able to mitigate these artifacts and improve image quality by better distributing the breast tissues, as shown in Fig. 33.The impact of the compression assembly on 3D ABUS acquisition and image quality was evaluated. The 3D ABUS acquisition performed without the compression assembly effectively removed any applied compression or pressure, while maintaining the natural curvature and structure of the breast. This approach could be especially useful for women who are lactating, where minimal compression is required when imaging sensitive structures and abnormalities in the breast53. Therefore, the 3D ABUS image has a high in-plane resolution  and poor out-of-plane resolution in the scanning direction, which is illustrated in Fig. 54. Another limitation is the current accessibility and adaptability of our prototype 3D ABUS design for widespread adoption in clinical practice, despite its cost-effectiveness and versatility. Adapting our SolidWorks CAD models to open-source programs would allow for improved dissemination for 3D ABUS design development, alteration, personalization for diverse patient populations, and optimization across various settings.We proposed a 3D ABUS approach, which included several design, technical, and potential clinical workflow advantages, demonstrated through the system’s fabrication, experimental validation, and healthy volunteer studies; however, there are some limitations in this study and areas for future work. Although the 3D ABUS system can accommodate any commercially available US transducer, thereby improving its cost-effectiveness and versatility, its 3D spatial resolution is limited in scanning direction due poor elevational resolution of conventional linear array US transducers, as determined by the elevational beamwidthWhile our study demonstrated adaptability of our design, geometric 3D reconstruction accuracy, 3D ABUS imaging capability in volunteer studies, and ability to mitigate motion artifacts, future extensive studies are still required to demonstrate its robustness across various diverse patient populations, including those with dense breasts. Prior to this exploration, further investigation is required to examine the robustness and adaptability of the proposed 3D ABUS system across a wider range of breast sizes and geometries that would be expected in clinical practice. Since this study only included healthy volunteer studies, its utility will be explored in a clinical feasibility study in comparison with supplemental HHUS, commercial ABUS systems, and screening mammography. Moreover, and of substantial importance, the overall impact that the proposed 3D ABUS system has on current clinical practices and workflow in breast cancer screening will be qualified in a future study. This includes an examination of the detection capability of our proposed 3D ABUS system, especially when compared to commercial systems used in the same context. With the reported results from this current study, a clinical feasibility with patients will be performed with a separate Research Ethics Board (REB) and Canadian regulatory  approval. Furthermore, its operator and device agnosticism will be evaluated to assess its ability to be implemented as a point-of-care screening approach in limited-resource or cost-constrained settings. This is especially important when considering environments with limited healthcare operator expertise, where high-quality image acquisition could provide reliable offline or remote assessment and interpretation for screening or diagnostic applications.We developed a cost-effective, portable, patient-dedicated 3D ABUS system for point-of-care breast cancer screening, which was fabricated with economical materials, can accommodate any commercially available US machine and transducer, and is personalizable to diverse patient populations. Our proposed system is especially advantageous due to its ability to provide full image coverage of the entire breast in a single 3D volume. Geometric reconstruction validation in phantoms demonstrated accurate linear and volumetric measurements, showing potential for quantitative measurements for screening and diagnostic evaluation. Demonstration of the 3D ABUS system and proposed workflow in three healthy volunteers showed utility for point-of-care 3D ABUS image acquisition, and whole-breast visualization and assessment. Importantly, the wearable assembly components enable improved tissue stabilization and comfort, adjustable compression, and mitigates potential acquisition artifacts due to patient motion and breathing since its frame of reference is affixed directly onto the patient. While future studies are required to evaluate its robustness across diverse patient populations and acquisition conditions, the 3D ABUS system shows potential utility for widespread adoption into clinical care for accurate, robust, and effective breast cancer screening applications, especially in increased-risk populations and limited-resource settings.


In the process of specimen deformation, unstable, metastable, and stable areas of plastic deformation localization are formed and replaced, and the formation of stable areas of localized plastic deformation, in which the specimen fails at the end of the test, can be detected at the initial stages of testing. In specimens, during the test in the zone of localized plastic deformation, bands are formed which pass through the entire surface of the specimen at an angle of 35 to 55 degrees to the tensile axis, and their development leads to the formation of stable zones of localized plastic deformation and to the failure of the specimen.This paper presents the results of uniaxial tensile tests on specimens of the hypoeutectic aluminum-silicon alloy A319. According to the results, the influence of surface treatment by pulsed electron beam on the mechanical properties of the material was determined. The peculiarities of deformation localization in the material caused by grinding of the surface layer material structure due to rapid crystallization during electron beam treatment were revealed. The surface treatment up to the depth of 100 µm leads to the formation of a fine dendritic columnar structure of silumin and to an increase in the plasticity of the samples. The influence of the surface treatment affects the increase in the deformation localization in the region of the stable concentrator before failure. The greatest increase in ductility and localization of deformation occurs during treatment with an energy density of 15 J/cm Changing the surface properties of structural materials can lead to an increase in the strength or plasticity of the product as a whole. Elimination of surface layer defects, refinement of grain structure, formation of optimal structural phase composition from the point of view of plastic deformation and fracture localization peculiarities can contribute to change the plastic deformation and fracture characteristics. High-energy pulsed electron beam is one of the promising tools for modifying the surface layer of metals and alloys . As a reA number of research groups are studying the behavior of the structure, defect substructure, elemental and phase composition ,5,6,7, iSeveral studies ,11 have In works ,12 it isA brief review of modern studies of the effect of electron beam treatment on the deformation behavior of metals allows us to conclude that this issue is a promising area of research and requires further development. At the same time, it is still unknown how a change in the structure and properties of a thin surface layer of the material can contribute to a change in the characteristic features of deformation localization and fracture of the samples as a whole. Therefore, the purpose of the present work is to study the influence of pulsed electron beam surface treatment of A319 grade hypoeutectic silumin on the characteristics of its plastic deformation and fracture under tensile stress.−1, electron beam energy density 15 and 25 J/cm2. As shown in [The samples were obtained from a hypoeutectic aluminum-silicon alloy of A319 grade (Al-(4-6)Si-1.3Fe-0.5Mn-0.5Ni-0.2Ti-2.3Cu-0.8Mg-1.5Zn, wt%). Samples in the form of bilateral proportional blades with the working area dimensions of 55 mm × 9.6 mm × 3.8 mm were prepared a. The saTensile testing of the specimens was performed on an Instron 3386 testing machine at a tensile speed of 0.2 mm per minute. The test scheme is shown in yy—along the Y axis, axis directed along the applied stress), transverse deformation  were determined and analyzed [Measurements of the development of deformation fields on the surface of the investigated specimens were carried out using the VicSnap program on the basis of the obtained synchronous recording of images from two cameras c. As a ranalyzed ,20. The Electron beam treatment of the surface causes rapid melting of the surface layer and subsequent crystallization at a sufficiently high rate. This results in the refinement of the alloy structure in the thin surface layer and the formation of a peculiar morphology on the surface. This, in turn, determines the characteristics of deformation and fracture of the material during testing. yy>  of untreated specimens and specimens treated with 15 and 25 J/cm2 electron beam energy density (ES).The plastic deformation of the specimens under test is fairly uniform until ultimate strength and fracture are reached. 2 increased by 55%, and with ES = 25 J/cm2 by 27%. The maximum relative strains of the untreated specimen were 0.234%, at ES = 25 J/cm2 were 0.297%, and at ES = 15 J/cm2 were 0.363%. The Young’s modulus of the untreated and electron beam-treated samples at ES = 25 J/cm2 is the same and is equal to 60 GPa, and at ES = 15 J/cm2 it decreases by 3.3% and is equal to 58 GPa. The tangent modulus of deformation at the point of failure of the specimen without treatment was 15 GPa. For the specimens treated with ES = 15 J/cm2 and 25 J/cm2, it decreased by 47 and 27%, respectively, which is equal to 8 and 11 GPa.Electron beam treatment of the surface does not result in significant changes in tensile strength. The maximum stress before failure of the specimens varies within 4%. However, analysis of the change in strain of the specimens during the test shows that the plasticity of the specimens changes quite significantly. Maximum relative strains of specimens treated by pulsed electron beam with energy density 15 J/cmThe increase in plasticity of samples treated by pulsed electron beam is caused by a significant change in the structure of the surface layer of silumin. The conducted studies show that in the cast state the structure of silumin A319 is represented by dendrites of solid solution based on aluminum, Al-Si eutectics, inclusions of silicon and needle-shaped intermetallides, which is clearly marked on the fracture surface a. The de2  was used to determine the overall deformed state of the specimen along the edges of the work area. Since the stress-strain diagram  over the sample surface (relative to the raw sample). The increase in plasticity (+27%) of silumin samples at electron beam treatment with energy density 25 J/cm2 due to both increased values of maximum longitudinal deformations <εyy> (+17%) and increased values of minimum deformations (+10%) across the sample surface (relative to the raw sample without treatment).The increase in plasticity (+55%) of silumin samples at electron beam treatment with energy density 15 J/cmxx> on a surface of silumin sample without treatment made 0.282%; at energy density ES = 15 J/cm2 values of deformations have increased on 89% and are equal to 0.535%; at energy density ES = 25 J/cm2 values of deformations have increased on 5% and are equal to 0.296%. The increase in ductility (+55%) of silumin specimens at electron beam treatment with energy density ES = 15 J/cm2 is due both to high values of maximum transverse strains <εxx> (+89%) along the specimen surface (in comparison with the starting specimen) and to increased values of minimum strains (+82%) along the specimen surface . The smaller increase in ductility (+27%) of silumin specimens at electron beam treatment with energy density ES = 25 J/cm2 is due to both smaller increase in values of maximum transverse deformations <εxx> (+5%) and smaller increase in values of minimum deformations (+23%) over the specimen surface (in comparison with the raw specimen).Values of maximum transverse deformations <ε2. However, the greatest ductility is characteristic of the specimens treated with an electron beam with an energy of 15 J/cm2.The conducted studies show that by treating the surface of hypoeutectic aluminum alloy A319 with pulsed electron beam it is possible to form in the surface layer a structure of the fine dendritic cell (columnar) type without inclusion of secondary phases of needle (lamellar) shape, which leads to a significant increase in plasticity of the material with a small change in strength properties. The depth of the treated zone varies from 15 to 100 μm. The greatest depth of the surface layer is characteristic of samples treated at 25 J/cm2, while the deformation bands are most pronounced in specimens treated with energy 25 J/cm2. Plastic deformation localization zones can be of stable, metastable, and unstable types. Zones of the first type form from small amounts of deformation and develop throughout the test. Zones of the second type may develop over a long period of time, but their effects then diminish or cease. Third type zones form for a short period of time and then disappear. The fracture structure of the base metal of treated and untreated specimens is represented by brittle chipping along the boundaries of the silicon inclusions. The destruction of the treated surface layer is characterized by intergranular fracture along the boundaries of columnar dendrite arms.The obtained data testify to the localization of deformation in all types of specimens during testing. This process begins with the formation of a chaotic or staggered distribution of localized areas of plastic deformation. Further development of the plastic flow leads to the formation of deformation bands from individual concentrators located at an angle of 45 degrees to the deformation axis. Bands of two orientations intersecting each other are present in the specimens. Areas of maximum localization of plastic deformation are formed at the intersection of such bands. The tendency to band formation increases after surface treatment with electron beam, and the deformation localization is maximum in specimens treated with electron beam of energy 15 J/cmThus, the results obtained demonstrate the effectiveness of electron beam surface treatment of aluminum-silicon alloys in reducing their brittleness and increasing their ductility.


Sitophilus granarius L.) is a major primary pest of stored cereals throughout the world. Among the major classes of plant secondary metabolites, flavonoids can affect insect feeding behaviour and their growth rate. In this study, the susceptibility of an anthocyanin-rich purple durum wheat genotype (T1303) to the granary weevil was evaluated in comparison with two yellow durum (Ofanto) and bread (Mec) wheat varieties. The feeding response and food utilisation efficiency by adult insects was also investigated by calculating nutritional indices in whole flour disk bioassays. Different levels of susceptibility to granary weevil emerged among genotypes tested. The mean food consumption by an insect, F1 progeny, and female parental offspring calculated for the T1303 genotype were significantly lower than those of yellow kernel wheat varieties. Moreover, T1303 genotype induced deterrence in the adult insects as demonstrated by the positive values of the food deterrence index. Besides, relative grow rate and efficiency conversion of ingested food indices were negative for T1303 and positive for both yellow wheat varieties indicating respectively a decrease and an increase of insect body weight during the bioassays. Finally, a higher mortality rate was recorded for insects fed on T1303 flour disks compared to disks obtained from yellow wheat varieties. These results provide evidence for the antifeedant and toxic effects of anthocyanins present in the T1303 pericarp against the granary weevil. Overall, this study contributes new insights into the mechanisms of host acceptance and food utilization by S. granarius and would be useful to identify antifeedant flavonoids as well as to develop varietal resistance-based strategies against this pest.The granary weevil ( Food security will depend on the ability to increase productivity while limiting losses during both cultivation2 and postharvest3. Insect pests during product storage are an increasingly global problem4. In fact, postharvest wheat losses, due to pest attacks, are estimated to be about 10–15% of the global annual production6 and, in some developing countries, they can reach 50% of the total harvest7.Wheat is one of the most important food crops to human populations consumed worldwideSitophilus granarius (L.) , is one of the most damaging primary pests of stored cereals worldwide. It can attack intact kernels and causes both severe quantitative and qualitative losses10 due to larvae and adults feeding and commodities contamination with exuviae, excrements and mycotoxins that may result from insect-promoted fungal growth during storage13.The granary weevil, 14. Thus, sustainable control means as alternatives to chemical inputs during cereal storage are urgently needed15. Possible alternative control methods include the use of botanicals powders, extracts, and essential oils (EOs) of plant origin23, semiochemicals24, inert powder31 and resistant varieties32.The endophytic development of immature stages, the stringent legislation on the use of synthetic pesticides and the increasing consumer demand for safer food make the control of granary weevil very difficult33. Volatile organic compounds (VOCs) play an important role in host finding because they are the first chemicals detected and used by insects to distinguish between suitable habitats and substrates and unsuitable ones37. The host-plant acceptance mainly depends on behavioural responses of insects to non-volatile plant chemical and physical features37.Food plant selection by phytophagous insects consists of food finding and food acceptanceSitophilus granarius adults are able to perceive a wide range of volatile compounds38 emitted by grains of several cereals40 and are attracted to the odour blend of commercial wheat with yellow kernels42. Moreover, plant secondary metabolites are involved in insect-plant interactions from habitat selection to host acceptance45.46 and are significant cues in host recognition and host acceptance47.Major class of secondary metabolites are flavonoids, including anthocyanins, which represent about 5–10% of the known secondary products in plants.48 found sitosterol and the flavonol isoquercitrin as biting factors for the silkworm, Bombyx mori L. Moreover, flavonoids could have insect feeding stimulant activity53. By contrast, many flavonoids are deterrents to insects, may be deleterious if ingested and detrimental to their growth44. Flavonoids have also been implicated as plant compounds that stimulate or deter the female oviposition44.Several studies showed the dual nature of flavonoids as insect feeding stimulants or deterrents. Hamamura et al54. In this context, pigmented wheat genotypes that carry purple genes controlling anthocyanin pigmentation on grain pericarp are very interesting57, due to the high nutritional value conferred to the final products59.The increasing interest in the health benefits of flavonoids, has prompted plant breeders to increment the levels of these compounds in cropsS. granarius did not exhibit a preferential orientation toward the odours of pigmented wheat genotypes during behavioural bioassays60.In a previous study, marked differences in VOCs profile emerged within pigmented and yellow kernel of durum and bread wheat genotypes and adults of S. granarius adults. To this end, intact kernel susceptibility tests and flour disks bioassays were carried out with two commercial yellow wheat varieties and a pigmented wheat genotype with a purple pericarp.The aim of the present study was to investigate the influence of wheat anthocyanin pigments in host acceptance and utilization by Sitophilus granarius were reared for several generations on whole durum wheat kernels (var. Simeto) in cylindrical glass containers (Ø 15 × 15 cm) covered with a fine mesh net (0.5 mm). Colonies were maintained in the dark at 25 ± 2 °C and 60 ± 5% relative humidity. Mixed sexes 30-day-old adult beetles were used for intact kernel susceptibility tests and flour disks bioassays.Triticum aestivum L. subsp. aestivum) variety “Mec” (Marzotto/Combine) and two durum wheat [(Triticum turgidum L. subsp. durum (Desf.)] genotypes: “Ofanto” (Adamello/Appulo), an élite variety with high grain yield and wide adaptability to the Mediterranean basin61, and a purple durum wheat genotype, “T1303” (USDA code PI 352395) with high levels of anthocyanins in the grain. The three wheat genotypes were grown simultaneously in a replicated (n = 10) field trial carried out at the CREA-CI of Foggia, Italy , on a clay-loam soil (Typic Chromoxerert) during the 2019–2020 growing season, using standard agronomic practices. The harvested wheat genotypes were analysed to determine the main qualitative and technological parameters and stored at low temperature (4 ± 1 °C) until needed for biological tests. Moisture of whole grains was determined using the single-stage air oven  method (ASAE 2003). This method utilizes whole grain dried for 18 h at 130 °C to determine the moisture content. Thousand-kernel weight (TKW) was calculated from the mean weight of three sets of 500 grains per each wheat genotype. Protein content (PC) was determined by nitrogen combustion analysis according to Approved Method 46–30  using a Dumas nitrogen analyzer . The Single Kernel Characterization System 4100 (SKCS)  was used to characterize kernels hardness (Ha) using a sample of 300 kernels (Method 55-31) . A grain sample with 800 g of each sample were also milled by a Bona 4RB mill  after tempering according to their hardness. The flour obtained was used to perform alveograph test, according to ICC-Standard no. 121 . The variables of alveograph deformation energy (W) and curve configuration ratio [P/L-relation between dough tenacity (P) and extensibility (L)] were measured.Three wheat genotypes were chosen for this study, including the bread wheat  were put into different tubes and diluted  with either potassium chloride buffer (0.03 M KCl), for pH 1.00, or sodium acetate buffer (0.4 M CH3CO2Na·3H2O), for pH 4.50. The resulting samples were incubated for 30 min at room temperature in the dark and then filtered with 0.45 μm regenerated cellulose syringe filters. The absorbances of the samples at 520 nm were measured against distilled water as the blank. Total anthocyanin content was corrected for the dry matter and is expressed as Cy-3-Glc equivalents as micrograms per gram of dry matter.Total anthocyanin content (TAC) was evaluated using a colorimetric method with different pH solutions as reported by Ficco et al.In order to ensure the absence of live insects inside the wheat kernels to be used for biological bioassays, samples were frozen at − 20 °C for 72 h before the experiments.S. granarius adults of mixed sexes. Containers were closed by screw caps and maintained in the incubator  at controlled conditions of photoperiod (L 0 : D 24), temperature (25 ± 2 °C), and relative humidity (60 ± 5%). For each wheat genotype there were 5 replicates. After 15 days exposure, insects were removed, sexed and the number of dead insects in each replicate was recorded. The F1 progeny was monitored by removing and counting newly emerged adults every 3 days. The experiment was terminated when no adults emerged for five consecutive days62.Susceptibility tests with intact kernels were performed on wheat samples conditioned for 7 days at 25 ± 2 °C, 60 ± 5% r.h. after frozen treatment. For each wheat genotype, not infested kernel samples (60 g) placed in cylindrical glass containers (Ø 9 × 14.5 cm) were infested with 12 63; (3) percentage of mortality during the oviposition period; (4) number of adult offspring per female; (5) percentage of weight loss = Wi–Wf/Wi × 100 were Wi = Initial dry weight and Wf = Final dry weight64; (6) food consumption by an insect.For each wheat genotype, the following parameters were calculated: (1) total number of F1 progeny; (2) median development period (D), estimated as the time, expressed in days, from the middle of the oviposition period to the emergence of 50% of the F1 generationFor each genotype, whole wheat flour was prepared by milling kernel samples (20 g) using a Tecator Cyclotec 1093  laboratory mill (1 mm screen-60 mesh). A sample (2.5 g) of each wheat flour was uniformly suspended in distilled water (8 mL) and stirred by a magnetic stirrer .65 that generated 60 ± 5% r.h.To obtain flour disks to be used in feeding bioassays, aliquots (200 μL) of suspension were dropped onto holes  of a rectangular support (15 cm × 15 cm) designed for this purpose and manufactured using a 3D printer . Then, the support was placed in a fume cupboard for 7 h until solid flour disks were obtained. Initial humidity of flour disks was stabilized overnight at 25 ± 2 °C in an airtight glass desiccator using a NaCl solutionIn a pre-weighed glass vial (22 mL) two flour disks and 5 group-weighed weevil adults were introduced. Each vial was then re-weighed using an analytical balance  and maintained in the incubator  at controlled conditions of photoperiod (L 0 : D 24), temperature (25 ± 2 °C), and relative humidity (60 ± 5%) for 6 days. For each genotype 5 replicates were set up. After 6 days, the glass vials with flour disks and alive insects were weighed again and the number of dead insects was recorded.67.Six days after the experiment start, the adult weevil mortality rate (%) was calculated. Moreover, the following nutritional indices were calculated: Relative Consumption Rate (RCR) = D/(B × day), where D = biomass ingested (mg)/No. of live insects on the sixth day; Relative Growth Rate (RGR) = (A − B)/(B × day), where A = mean weight (mg) of live insects on the sixth day, B = initial mean weight (mg) of insects; Efficiency Conversion of Ingested Food (ECI) = (RGR/RCR) × 100; Feeding Deterrence Index (FDI) (%) = [(C − T)/C] × 100, where C = consumption of control disks (Mec or Ofanto) and T = consumption of disks of the genotype considered (T1303)Data were submitted to analysis of variance (ANOVA) followed by Tukey’s HSD test for mean comparisons. Before ANOVA, data were submitted to Shapiro–Wilk’s test to verify the normal distribution of data and to Levene’s test to assess the homogeneity of variances. Statistical analyses were performed with SPSS  v.18 for Windows .Plant materials used in the present study are compliant with the local and national guidelines.This article does not contain any studies with human participants or animals performed by any of the authors.Informed consent was obtained from all individual participants included in the study.df = 2 P = 0.019; PC: F = 19.397 df = 2 P = 0.002; Ha: F = 3309.80 df = 2 P = 0.000; W: F = 1393.00 df = 2 P = 0.000; P/L: F = 28.231 df = 2 P = 0.000). The commercial durum wheat variety (Ofanto) showed the highest TKW and Ha values but its PC (13.5 ± 0.5%) was significantly lower than those of the other genotypes. The bread wheat variety Mec showed the lowest values of Ha, being a variety with medium/soft kernels, and the most suitable alveographic parameters for making bread . The T1303 genotype was the only one with the presence of anthocyanins in the grain (40.2 ± 0.03 mg/kg), a high protein content (15.4 ± 0.8%), similar to Mec (16.1 ± 0.5%), and a Ha value (85 ± 3) similar to that of the Ofanto variety (88 ± 3).The average values of the main qualitative and technological parameters determined on the wheat kernel samples immediately after harvesting are reported in Table S. granarius adults kept on the purple kernel genotype T1303 (16.7 ± 4.8%) was higher than those observed for the yellow wheat varieties, even if not significantly different  . The mean number of adults emerged from T1303 (67.0 ± 11.9) kernel samples was significantly lower than those obtained from Mec (213.7 ± 27.0)  and Ofanto (213.0 ± 6.1)  samples. This resulted in significant differences  among the offspring originated by one parental female on different wheat genotypes (Table S. granarius emerged from the purple wheat genotype T1303 (42.1 ± 0.7) was the highest but not significantly different  compared to the yellow Mec (41.0 ± 0.6) and Ofanto (40.7 ± 0.4) varieties  among different genotypes (Table P < 0.05) than those observed for Mec (6.75 ± 0.31%) and Ofanto (7.02 ± 0.04%) varieties   than those recorded for Mec (19.37 ± 1.57 mg) and Ofanto (19.82 ± 0.71 mg) varieties   was lower than that of insects fed on disks of yellow wheat varieties, even if not significantly different . The mean RGR on flour disks from the T1303 purple genotype was negative (− 0.025 ± 0.004 mg/mg/day) and significantly lower  than the positive RGR calculated for Mec and Ofanto  varieties  than those of Mec (30.7 ± 4.2) and Ofanto (36.9 ± 6.2) flour disks   mean mortality rate (24.0 ± 4.0) was induced in insects fed on T1303 flour disks  and Spodoptera frugiperda (JE Smith), suggesting their suitability as biopesticides79. By contrast, another study suggested that there are no differences in susceptibility to insect attacks during storage between white and red wheat varieties80.Early studies showed that polyphenol-rich pericarp purple corn extracts, containing anthocyanins, have several negative effects on the growth, development and fitness of different stages of In the present study, susceptibility bioassays revealed a significant reduction in the total number of F1 progeny and female parental offspring emerged from purple wheat genotype compared with yellow wheat varieties. Moreover, insects fed on pigmented grains consumed less food substrate than those reared on yellow varieties, which resulted in a significantly lower damage. These results strongly suggested a different level of susceptibility among the purple genotype and yellow varieties studied.82 and content of some chemicals, such as protein or gluten, total lipids and cuticular lipids88 that are function of genetic and environmental factors90. Besides, the role of pericarp cell wall components in cereal weevil resistance has also been one of the aspects studied in the past on various cereals92. However, several studies have shown that the different varietal response to external stress was much more likely caused by complex interactions between structural factors (proteins and polysaccharides) and phytochemicals present in the grain96.It is known that host acceptance by stored cereal pests depends on both physical and chemical properties of grain kernels. Indeed, the susceptibility of various wheat cultivars to insect pests has been related to physical kernel features, such as water content, hardness and diameter of kernel, thousand-kernel weight, vitreosityIn our study, anthocyanins were contained exclusively in the pigmented genotype T1303 whereas qualitative and technological parameters of T1303 were similar to those of at least one of the two yellow wheat varieties. In fact, the pigmented T1303 genotype showed a thousand-kernel weight value and protein content similar to the yellow Mec variety and hardness values similar to the Ofanto variety.S. granarius among yellow and purple kernel genotypes appeared to be associated with differences in the content of phenolic compounds, particularly anthocyanins. This is consistent with Kordan et al.84, which showed that a greater presence of phenolics in wheat grain determines a higher adult mortality and a reduction of insect fitness and damage.In this contest, marked differences registered in population dynamics of S. granarius, flour disk bioassays, using whole flour of each genotype, were set up to definitively overcome the influence of physical features.Thus, considering the possible role of pericarp anthocyanins in the susceptibility of T1303, Mec and Ofanto to S. granarius adults fed on T1303 wheat flour disks were significantly higher than those observed for insects fed on yellow wheat varieties.However, although small differences in the amounts of flour disk ingested, different food conversion efficiency were found among the purple and yellow genotypes. Indeed, RGR and ECI values were positive for the Mec and Ofanto varieties and negative for T1303 indicating respectively an increase and a decrease of insect body weight during the experiments. Besides, the positive FDI values calculated for T1303 using Mec or Ofanto as controls indicated actual feeding deterrence of the T1303 genotype. Lastly, after six-day exposure, the mortality percentages of 96.On the whole, our results strongly suggested that the anthocyanins accumulated in the pericarp of T1303 kernels, evenly distributed in the whole flour used for flour disks preparation, determined antifeedant, deterrent and toxic effects against granary weevil adults that may explain the differences in susceptibility observed. Certainly, a more thorough investigation will have to be conducted on other plant’ secondary metabolites that could interfere with the metabolism of the insectIn this context, our results pave the way to better understand the biological activity of the phenolic fraction of T1303 pericarp extracts and to identify their bioactive components. Finally, flour disk bioassays appear very promising for further wheat genetic investigation because they could be well suited to high-throughput analyses required by -omics approaches and, to accelerate the transfer of genes or genetic regions associated with resistance in a modern breeding program through marker-assisted selection. From a practical point of view, our results strongly suggest that purple wheat genotypes could be exploited in breeding programs to improve wheat resistance to the attacks of post-harvest stored pests, contributing to the alternative control options.


NLR gene clusters have recently gained momentum owing to their robustness and malleability in adapting to recognize pathogens. The modular domain architecture of an NLR provides valuable clues about its arms race with pathogens. Additionally, plant NLRs have undergone functional specialization to have either one of the following roles: to sense pathogen effectors (sensor NLRs) or co-ordinate immune signaling (helper or executer NLRs). Sensor NLRs directly recognize effectors whilst helper NLRs act as signaling hubs for more than one sensor NLR to transduce the effector recognition into a successful plant immune response. Furthermore, sensor NLRs can use guard, decoy, or integrated decoy models to recognize effectors directly or indirectly. Thus, by studying a plant host’s NLR repertoire, inferences can be made about a host’s evolutionary history and defense potential which allows scientists to understand and exploit the molecular basis of resistance in a plant host. This review provides a snapshot of the structural and biochemical properties of the different classes of NLRs which allow them to perceive pathogen effectors and contextualize these findings by discussing the activation mechanisms of these NLR resistosomes during plant defense. We also summarize future directives on applications of this NLR structural biology. To our knowledge, this review is the first to collate all vast defense properties of NLRs which make them valuable candidates for study in applied plant biotechnology.Plants possess an arsenal of immune receptors to allow for numerous tiers of defense against pathogen attack. These immune receptors can be located either in the nucleocytoplasm or on the plant cell surface.  Coiled-coil (CC)-NLRs (CNLs), 3. Resistance to powdery Mildew 8 (RPW8-like) CC-NLRs (RNLs) (s (RNLs)  and mores (RNLs)  of whichs (RNLs) . The afos (RNLs) . Additios (RNLs) .R gene and its corresponding effector (avirulence gene (Avr)) through the gene for gene model. Although Arabidopsis CNL called Suppressor of MKK1 MKK2 2 (SUMM2), provides a well-studied example  Figure 2G10 domain which allows categorization into their respective groups (OG3 domain comprising of a Met-Ala-Glu-Pro-Leu (MAEPL) amino acid motif, is commonly found in non-flowering plant lineages, whilst the MADA and MADA-like motifs  are found within angiosperms  which acts as a downstream signaling molecule in plant defense , a helical domain 1 (HD1) and a winged helix domain (WHD) . The NBDPm3) gene possesses an unusually large LRR domain which has been classified as an “island domain” which forms extending loops on the exterior of the canonical LRR domain  and hydrophilic amino acid residues (LxxLxLx) . These sR domain . The LRRR domain .Arabidopsis NLR RPS4 containing a functional N-terminal TIR domain but lacking the NB-LRR domain has been demonstrated to trigger immunity , WRKY and heavy metal associated domain (HMA)  . Althougin (HMA) . A compain (HMA) . Specieshared ID . This alhypodium . This fr2.2Arabidopsis NLRs, RRS1/RPS4 have been shown to recognize the bacterial effectors AvrRps and PopP2 from P. syringae pv. pisi and Ralstonia solanacearum, respectively through an integrated WRKY domain in the C-terminal region of RRS1 (WRKY domain is hypothesized to mimic the DNA-binding domains in plant WRKY transcription factors (TFs) involved in activation of defense genes  are metal ion binding chaperone proteins which possess a characteristic isoprenylation motif and a heavy metal associated (HMA) domain . The paiAvr-PikD . It was Avr-PikD . MaidmenArabidopsis trithorax1 (RATX) domain at the C-terminal region located after the LRR domain. Alternative splicing of RGA5 generates two different isoforms of RGA5, namely RGA5-A which contains the HMA domain and RGA5-B which lacks the HMA domain  sensors which potentially facilitate the ZAR1 mediated effector recognition of the Hop family of effectors from P. syringae  domain which contributes to effector recognition     proteins act as redundant helper NLRs to work with numerous sensor NLRs . This lent hosts . In the nt hosts .Arabidopsis, the RRS1/RPS4 pair was shown to require ADR1 for complete resistance  lacking ADR1 genes showed the former to exhibit a more susceptible phenotype (ADR1 and NRG1 genes work in tandem rather than interchangeably to contribute to full resistance. The RRS1 sensor NLR in the RRS1/RPS4 pair was previously characterized as an NLR-ID owing to the presence of the WRKY motif (The second type of NLR network that has been characterized is the N Requirement gene 1 and Activated Disease Resistance 1 family (NRG1/ADR1). This is an RNL type of helper NLR family required to act as helper NLRs for all TIR NLR sensors. Studies have shown a tight correlation of copy number between TIR-NLRs and RNLs like NRG1 thereby substantiating a signaling link between the two families . Sensor sistance . Howeverhenotype . This shKY motif . It is u2+ channels  and necrosis. It is proposed that PCD is a broad concept encompassing two classes. The first class is defined as vascular cell death whilst the second class is defined as necrotic cell death . Class o2+ signaling  network likely forms a resistosome structure mimicking that of ZAR1 to co-ordinate immune signaling. All TIR-NLRs transduce effector recognition into an immune response using the NRG1/ADR1 network, both of which are RNLs. This pathway shows remnants that are very similar to those found in animals and prokaryotes. There are two ways in which TIR-NLRs enzymatically initiate a HR upon effector recognition. In the first mode of action, a sensor TNL is activated via effector interaction to form a TNL resistosome (+ to create v-cADPR products which incorporate into a family of lipase-like proteins comprising of Enhanced Disease Susceptibility 1 (EDS1), Phytoalexin-Deficient 4 (PAD4) and Senescence Associated Gene 101 (SAG101)    and demonstrated increased resistance to two potato virus X strains (CPTK and CPKR) . Owing t capsici . These mi (I141N . This I2opersici . Thus, tffectors .M. oryzae pathosystem, showed how polymorphism amongst NLRs facilitated the recognition of different effector alleles (Pikm alleles have been demonstrated to have the ability to recognize any of the Avr-Pik effector variants, owing to the presence of the HMA domain (Pikp allele can only recognize one of these variants, assumably due to the lack of the HMA domain which acts as the site of polymorphism. However, M. oryzae effector Avr-PikD whilst RGA5 recognizes the effectors Avr-Pia and Avr1-CO39. Effector binding residues from the HMA domain in Pikp-1 were introduced into the HMA domain in RGA5 to create two mutants, RGA5_HMAm1 and RGA5_HMAm2. These effector binding residues were determined from prior structural studies of HMA-effector interactions (N. benthamiana illustrated that RGA5 variants carrying this engineered domain recognize a new effector, Avr-PikD in addition to their original effectors.Other studies have engaged in rational NLR design using guard, decoy or integrated decoy NLR systems. Some modifications include the editing of IDs or the swapping out of IDs between NLRs or swapping cleavage sites within plant proteins to modify the activation of guard or decoy systems in response to wider pathogen stimuli. A proof-of-concept study in the Pik-1/Pik-2 system in the rice- alleles . In thisA domain . In contractions . Co-exprArabidopsis operates using a guard model where the RPS5 NLR is activated upon P. syringae AvrPphB effector mediated cleavage of the plant protease PBS1 (The RPS5/PBS1 system in ase PBS1 . Studiesase PBS1 . The repase PBS1 . The samase PBS1 . Using tase PBS1 . PottingSeveral studies have sought to conduct natural variation analysis to identify NLR variants possessing mutations which confer higher fitness to the host via effector recognition . One stuNLR alleles dictate protein conformations which favor activation. A study conducted in flax NLRs demonstrated the structural basis of two receptors, L6 and a weaker counterpart, L7 (Mutations within part, L7 . Using spart, L7 . This coNLRs are activated via the release of the auto-inhibition state. Therefore, by inducing mutations that promote this release, NLRs with expanded recognition spectra can be engineered . In the Other avenues of research have looked to conduct cross kingdom studies of TIR domain functions to improve TNLs’ functions against plant pathogens. Under normal conditions, an insect-transmitted phytoplasma effector known as SAP05 targets a family of GATA zinc finger transcription proteins . One stuM. oryzae could not be observed when introduced into rice (Despite research being directed towards the rational design of NLR receptors, some caveats still exist. Although the engineered rice NLR mutants RGA5_HMAm1 and RGA5_HMAm2 show extended recognition spectra in model plant species, this extended resistance against nto rice . Both munto rice , hence tAlthough the past few years have culminated vital proof-of-concept research pertaining to engineered NLR receptors, some challenges remain to be tackled. A few studies have indicated that the induction of mutations or domain swapping can cause auto-immune phenotypes when transiently expressed in heterologous model systems . The cre5NLR genes is not ubiquitous across all food cultivars. Papaya, watermelon, and cucumber are a few crops possessing a low number of NLRs (This review has coalesced vital research pertaining to NLR structural biology to contextualize the larger picture of how NLRs operate in immune signaling pathways. This has opened the Pandora’s box of research to exploit these properties to harness NLR defense potential into a universal defense model applicable to broader systems. Research has been directed towards retooling NLR pathways to create an adaptive immune system mimicking that of higher mammals. Two main reasons have been postulated to drive research towards the creation of made-to-order NLRs. Firstly, the presence of  of NLRs . This ha of NLRs . This po of NLRs .The impetus for Another study was able to restore NLR activity previously nullified by a pathogen effector by introducing core mutations which would allow it to surpass deactivation by the effector . An effeIt is highly likely that more studies will pave the way to generate plant antibodies using NLRs as a framework or work to integrate the structural basis of NLR-effector recognition to restabilize NLRs nullified by pathogen effectors. Embarking on such NLR engineering studies should be underscored by analyzing promising NLR candidates in stable transgenic lines to better understand their durability and to test the potential of deleterious phenotypes that could arise from overexpressing NLRs. Although the engineering of NLRs provide promise in supplying bespoke, broad-spectrum resistance in plants, the issue of durability and transmissibility needs to be addressed. Such novel immune receptors should be cautiously deployed into crops to ensure that they are not nullified by the adaptive potential of plant pathogens.SA: Conceptualization, Writing – original draft, Writing – review & editing, Investigation. VS: Supervision, Writing – review & editing, Conceptualization. NB: Funding acquisition, Supervision, Writing – review & editing, Conceptualization.


TCF4 haploinsufficiency by deletions, truncating variants or loss‐of‐function missense variants within the DNA‐binding and protein interacting bHLH domain causes Pitt‐Hopkins syndrome (PTHS). This neurodevelopmental disorder (NDD) is characterized by severe intellectual disability (ID), epilepsy, hyperbreathing and a typical facial gestalt. Only few aberrations of the N‐terminus of TCF4 were associated with milder or atypical phenotypes. By personal communication and searching databases we assembled six cases with the novel, recurrent, de novo missense variant c.1165C > T, p.(Arg389Cys) in TCF4. This variant was identified by diagnostic exome or panel sequencing and is located upstream of the bHLH domain. All six individuals presented with moderate to severe ID with language impairment. Microcephaly occurred in two individuals, epilepsy only in one, and no breathing anomalies or myopia were reported. Facial gestalt showed some aspects of PTHS but was rather non‐specific in most individuals. Interestingly, the variant is located within the AD2 activation domain next to a highly conserved coactivator‐recruitment motif and might alter interaction with coactivator proteins independently from the bHLH domain. Our findings of a recurrent missense variant outside the bHLH domain in six individuals with an ID phenotype overlapping with but not typical for PTHS delineate a novel genotype–phenotype correlation for TCF4‐related NDDs.  About 20% of causative variants are missense variants located in the highly conserved bHLH domain, encoded by exon 18  pathogenic de novo in five individuals) upstream of the bHLH domain and presenting with rather non‐specific moderate to severe ID without or with only few facial aspects of PTHS.We now report on six individuals harboring a recurrent missense variant (confirmed to be 2TCF4 (NM_003199.3 or NM_001083962). The variant occurred de novo in five individuals. In one case, the father was not available. Consent to publish clinical and molecular data with or without clinical photographs was obtained from the parents or legal representatives.By personal communication among colleagues, being contacted by one of the families, and by searching ClinVar,in silico predictors .Genetic analysis was performed by either diagnostic trio exome sequencing in three cases or sequencing clinical exome or panels of 56 or nine ID genes, respectively, in the others Table . The p. missense variant in As reflected by three clinical scoring systems, the most distinctive clinical aspect of PTHS is the recognizable facial gestalt.TCF4, encoded by exon 18. This domain is crucial to form homodimers or heterodimers with other bHLH or HLH transcriptions factors and to bind DNA. It has been demonstrated that missense variants in the bHLH domain result in decreased transcriptional activation,PTHS‐causing missense variants are usually located within the bHLH domain of de novo c.1073G > T, p. variant in exon 14 was identified in a 20‐year‐old individual within a PTHS cohort, presenting with severe ID, breathing anomalies, constipation, but without seizures and with a less typical facial gestalt.de novo missense variant, c.1604A > G, p.(Asp535Gly) in exon 17 was also identified within a PTHS cohort but clinical details were not provided.in vitro but to change the ASCL1 and TCF4 heterodimer versus TCF4 homodimer binding preference.in vitro assays.Only few missense variants outside the bHLH domain of TCF4 have been reported. The Consequently, in at least three of the six individuals, the c.1165C > T, p.(Arg389Cys) variant was initially classified as of unknown significance due to its location outside the bHLH domain and to a rather unspecific neurodevelopmental phenotype not suggestive for PTHS. Only the recurrence of the identical variant in several individuals allowed its reclassification as pathogenic. This underlines the importance of careful interpretation and clinical follow‐up of exome sequencing results.Tcf4 truncation (Tcf4tr/+) mouse model which displayed hyperventilation and apnoea.Interestingly, the c.1165C > T, p.(Arg389Cys) variant is located in the AD2 activation domain next to a highly conserved Φ‐x‐x‐Φ‐Φ motif (amino acid residues 393–397), which consists of three hydrophobic amino acids (Φ) that are spaced by two variable amino acids (x). This motif is conserved between TCF4 and TCF12 and mediates binding to the KIX domain of the transcriptional co‐activator CBP/p300.tion Tcf4tr/+ mousTCF4‐related NDDs.To conclude, our findings of a recurrent missense variant upstream of the bHLH domain in six individuals with rather non‐specific, severe ID overlapping with but not typical for PTHS delineates a novel genotype–phenotype correlation for Bernt Popp, Thierry Bienvenu, Irina Giurgea, Julia Metreau, Cornelia Kraus, André Reis, Jan Fischer, María Palomares Bralo, Jair Tenorio Castano, Pablo Lapunzina, Berta Almoguera, Fermina Lopez‐Grondona, and Christiane Zweier collected mutational and clinical data. Heinrich Sticht performed structural modeling. Bernt Popp and Christiane Zweier wrote the manuscript, which was read and revised by all co‐authors.The authors declare no conflict of interest.