Low Plate Research Articles

Development of a low energy loss micro-channel plate based position-sensitive detector for the Rare Radio-Isotope Ring

A position-sensitive micro-channel plate type detector has been developed for use in the Rare Radio-Isotope Ring at RIKEN, Japan. It uses secondary electrons emitted from a conversion foil and deflected at 90° to create a position signal. Design constraints included operation in a high-vacuum, a large area to cover the ring acceptance and low beam interaction to reduce energy loss. It must achieve a precision sufficient to measure the in-ring dispersion and perform position diagnostics for tuning the injection. Reducing the time-of-flight of secondary electrons was principal in improving resolution as it reduces their final Gaussian spread. This was implemented by compacting the geometry of the detector and raising the acceleration potential. Electric field homogeneity was also improved by decreasing the electrostatic grid wire pitch from 2 mm to 1 mm. Offline tests with alpha sources and online tests with a 200 MeV/u 84Kr beam were conducted, reaching a final average position resolution of σ = 1.3 mm. This is sufficient for conducting beam diagnostics in the storage ring.

Microcavity-assisted cloning (MAC) of hard-to-clone HepG2 cell lines: cloning made easy

Cloning is a key molecular biology procedure for obtaining a genetically homogenous population of organisms or cell lines. It requires the expansion of new cell populations starting from single genetically modified cells. Despite the technical progress, cloning of many cell lines remains difficult. Cloning often fails either due to the strenuous conditions associated with manipulating cells or because many cells don’t tolerate a single-cell state. Here we describe a new cloning method utilizing low adhesion microcavity plates. This new technique, named microcavity-assisted cloning (MAC) was developed to clone difficult-to-clone HepG2 cells. The clones were produced following CRISPR/Cas9 knockout of the GSTA1 gene by a random distribution of 200, 400, and 800 cells into 550 microcavities of a 24-well low adhesion plate originally designed for the culture of spheroids. The knockout of GSTA1 was verified at the protein level using Western blotting. The advantages of the MAC method are its low cost, ease of the procedure, and the possibility of scaling up the throughput and automatization.

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of <12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth. Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 °C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3(451 µm x 462.84 µm)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Food Production in Ergonomically Designed Unit with Low Energy Consuming Thick Film Hot Plate

The Trans-Himalayan region of Ladakh is a cold desert where the temperature drops as low as -45°C, which makes the cultivation of fresh food almost impossible. The region is strategically very important as it borders two countries China and Pakistan. The unavailability of fresh food causes nutritional deficiency in the deployed troops and locals, affecting their performance. Cultivation of fresh food is very difficult due to the average low temperature and mountainous soil of the region. Transportation of fresh food to such forward locations causes decaying of nutrition besides the difficulty of reaching such snow-bound mountainous locations. Nutritious micro-greens grown in media under controlled conditions look perfect solutions for meeting nutritional requirements. The present study focuses on year-round cultivation of nutritious micro-green (2-3 times more nutritious than mature counterparts) with low energy consuming thick-film hot plate for necessary temperature maintenance in properly designed portable structures to reduce dependency on packaged food which are the causes of many diseases. The study involves the development of a thick film energy-efficient micro-climate maintenance technology-based system for the maintenance of temperature along with phase-wise development of suitable cultivation structure and standardization of the number of such plates and sizing of the portable unit.These hotplates produce heat based on the principle of Joule heating or resistive heating and the power of a hotplate is equal to I2R. The hotplates are capable of raising the surface temperature to 90°C –100°C at the expense of only 8W power consumption and maintenance of average temperature in the range of 18°C to 20°C at -25°C to -40°C outside ambient temperature. The region receives the highest solar irradiance along with annual average of 7-8 hours of sunshine duration and hence a solar-based technology can fulfill the energy supply of 8W with ease.The study revealed that a suitable temperature can be maintained for growing variety of nutritious micro-green in a triple layer-based multitier structure integrated with low energy consuming (~8 W), small (50.8 mm (x) 50.8 mm), efficient, and lightweight thick film technology.Thick-film hot plate integrated standardized unit is capable of producing an average of 1000grams/trial/unit in a short period of 8-12 days during the winter period of 6-8 months for the targeted supply of 8-10grams/person/day with total energy consumption of approximately 20-25Kw/trial at ~3350 m to ~5400 m Altitude.

Induction assisted autogenous plasma arc welding of HSLA steel

In this work, a high frequency induction heating (HFIH) assisted plasma arc welding (IPAW) technique is proposed to weld 6 mm thick S690QL high strength low alloy steel plates in square butt joint configuration and without using any filler material. A 3D finite element based coupled electromagnetic-thermal analysis is carried out to ascertain the weld's thermal characteristics. Microstructure evolution and mechanical performance are investigated using scanning electron microscopy, electron back scattered diffraction, transmission electron microscopy, tensile test, Charpy impact test and micro hardness test. The results demonstrate that 15 s initial static heating using a co-directional current coil with magnetic flux concentrator predominantly improves the weld penetration by 48 % and joint efficiency reaches 90 % to the base plate strength. Microstructural study shows that the addition of HFIH promotes low angle grain boundaries (LAGB) with bainite ferrite and granular bainite micro-constituents in the fusion zone, which causes localised yielding and failure in this zone during tensile test. Further investigation reveals that the HFIH encourages the formation of BI-type bainite ferrite laths of 1.3–2.2 μm width, combined with slender martensite-austenite (M-A) island at the bainite ferrite lath boundaries. The results also reveal that the induction heating promotes the formation of M23C6 precipitates and B2 structured Cu-enrich nano precipitates in the fusion zone (FZ). The microhardness distribution indicates that the FZ and coarse grain heat affected zone are significantly reduced due to the assistance of the HFIH. The impact test result shows a lower energy absorption by the IPAW weldments due to dominance of the LAGB with unfavourable strain distribution.

Effects of intercritical annealing time on microstructure, tensile properties, and cryogenic toughness of newly designed ultra-low C low Ni Medium-Mn steel

An ultra-low carbon and low nickel medium manganese heavy steel plates was fabricated by quenching and intercritical annealing process. The effect of annealing time on the tensile properties and impact toughness were studied in relation to the formation of reversed transformation. The results show that excellent mechanical properties with yield strength of 680 MPa, tensile strength of 871 MPa, total elongation of 38.2% and high impact energy of 187 J at −60 °C were obtained. The microstructure comprised of ultra-fine grained ferrite and retained austenite (RA) together with a small amount of martensite after intercritical annealing. Increasing the intercritical annealing time resulted in coarser matrix microstructures, larger and more voluminous retained austenite, but with notably reduced stability. Highly stable RA enhances the work hardening capacity of the steel via sustained deformation-induced transformation (TRIP effect), significantly contributing to the toughening of the multiphase system. In addition, Austenite stability, rather than large-angle grain boundaries and the amount of austenite transformation, is the primary factor determining the low-temperature toughness of medium manganese steel.

Experimental study on single value fluctuation of impact work in 700 MPa low-alloy tempering plate

The single value fluctuation of impact work occurs frequently in high strength steel, especially for medium or super thick plates. With respect to the single value fluctuation of impact work in 700 MPa low alloy thin tempered plate, the present study carried out a comprehensive and comparative analysis through metallographic microstructure, second-phase precipitated particle and inclusion distribution. The results show that the microstructure type, grain boundary ratio of HAGBs/LAGBs, grain size and orientation of the specimens with 37 J and 7 J impact work are basically the same. The two specimens show excellent process-microstructure consistency, which fully indicate that the temperature fields of the two specimens are consistent. Since the temperature field is basically the same, the (Ti, Nb, Mo)C compound precipitated particles in the two specimens maintain a good Baker-Nutting relationship with the matrix, and the interface between the precipitated particles and the matrix maintain a stable coherent/semi-coherent relationship. The main reason for the decrease of impact work of tempered plate is related to the distribution of inclusions. There are more spherical and square large size inclusion particles and microcracks in the specimen with lower impact work. Among them, the large square inclusion particles dominate mostly. And with the increase of the plastic deformation degree of the metal matrix, the large square inclusion has an obvious evolution by separation from the matrix to forming microcracks, and then to fracture.

Influence of the carbon fiber plate curvature of advanced footwear technology on the running energetic cost and 3000-m performance

This study aimed to explore the effect of two similar commercialized advanced footwear technology models differing mainly in their plate geometry (i.e., low and high curvature plate) on the running energetic cost in both non-fatigued and fatigued states. It also sought to determine their impacts on 3000-m performance. Twelve highly trained male athletes performed four 5-min trials (two advanced technology models, two trials). In each experimental session, athletes completed a 3000-m trial which was followed by a 5-min trial to test the running energetic cost when fatigued. Athletes exhibited a lower running energetic cost when wearing the low curvature plate in a non-fatigued state (P = 0.034; − 1.13 [− 1.92 to − 0.11] %), which persisted with better performance in the 3000-m trial (P = 0.017; − 0.42 [− 0.74 to − 0.09] %). However, in a fatigued state, non-significant differences were reported (P = 0.207). The low curvature plate seems to be more effective than the high curvature plate when running at low intensity in a fresh state as well as in a maximal high-intensity effort. However, non-significant differences between plate geometries were observed when fatigue was increased.

Anatomical study of the safety corridor for bridge plating positioned on the lateral border of the humerus.

This study shows the danger zone and the safety corridor in the lateral approach with bridge plating by measuring the distance between the lateral side of the plate positioned on the lateral aspect of the humerus and the radial nerve after it pierces the lateral intermuscular septum, in the different forearm positions. Forty arms of 20 human cadavers were used, the radial nerve was identified and marked on the lateral surface the radial nerve at the exit of the lateral intermuscular septum and anteriorisation of the nerve in relation to the humeral shaft and the lateral epicondyle was also marked. The distances were measured with a digital caliper. A submuscular extraperiosteal corridor was created, proximally between the biceps brachialis and deltoid muscle and distally between the triceps and brachioradialis muscle, followed by the positioning of the low contact large fragments contoured plate with 14 combined holes (fixed and cortical angle), inserted from distal to proximal. Measurements were performed in four positions (elbow flexion with forearm pronation, elbow flexion with forearm supination, elbow extension with forearm pronation and elbow extension with forearm supination). Significant statistical differences occurred with the different positions, and the elbow flexion with forearm supination was shown to be the position that provides the safest submuscular extraperiosteal corridor in a lateral approach of the humerus. The danger zone of radial nerve is an area that extends from 15cm to 5cm proximal to the lateral epicondyle and the safest way to create a submuscular and extraperiosteal corridor in the lateral region of the humerus is with the elbow in flexion and the forearm in supination.

Research on the welding process of Q355ND thick plate with narrow gap double wire submerged arc welding

Abstract Aiming at the problems of low welding efficiency and unstable quality of thick plates of marine equipment in offshore deep-water areas represented by offshore wind power, the high-efficiency welding process of narrow-gap double-wire submerged arc welding of 30 mm thick Q355ND low alloy high-strength steel plate was developed by selecting the suitable welding material and welding process parameters. The results show that the welded joint has a beautiful appearance, well-fused sides, and no defects. The structure of the heat-affected zone of the joint is mainly composed of pro eutectoid ferrite, acicular ferrite, bainite, and a small amount of pearlite. The center of the seam is composed of a large amount of massive ferrite and a small amount of bainite. The average tensile strength of the joint reaches 536.5 Mpa, and the average impact energy of the weld center at -20°C reaches 48.4 J. The average impact energy of the heat-affected zone reaches 164 J. All physical and chemical performance tests meet the standard requirements. The development of welding processes has important practical significance for improving production efficiency and product quality. It will be widely used to construct offshore wind power towers, offshore platform jackets, and other offshore deep-water engineering equipment.

Enhancing the mechanical and magnetic properties for low magnetic stainless-steel plates by non-recrystallization hot rolling and solution treatment

Enhancing the mechanical and magnetic properties for low magnetic stainless-steel plates by non-recrystallization hot rolling and solution treatment

A Review on Slit Steel Shear Walls: Development, Implementation, and Performance

Over centuries, natural phenomena have claimed a large number of lives and caused large amounts of damage. However, they were the reason for the development of knowledge and science. In engineering, there is no doubt that earthquakes were the driving force behind many design philosophies and advanced technologies. In this regard, steel plate shear walls (SPSWs) represent one of the technologies employed in both new constructions and existing structures to bolster lateral load resistance. In addition to its high elastic stiffness and strength, SPSWs often experience significant pinching during their hysteretic response unless heavily stiffened. Thus, numerous investigations have been performed recently to enhance the seismic behavior of SPSWs during severe ground shaking. The shear walls of steel plates have been studied in various ways, including stiffened and unstiffened steel plates, low yield strength steel plates, SPSWs perforated with circular holes or vertical slits, steel walls with stiffened and unstiffened openings. A stiffened SPSW panel dissipates significantly more energy than an unstiffened panel, as well as exhibiting a ductile and stable behavior. In view of its high elongation, the SPSW with low yield point has the best damping capacity. It has been demonstrated that steel walls perforated with circular holes have improved energy dissipation, in addition to allowing utilities to pass through their openings. Vertical slits on steel plate shear walls produce an exceptionally full hysteresis loop with improved performances. Slit steel walls (SSWs) can sustain a drift of over 3% while experiencing minimal hysteresis degradation. Additionally, a frame with a SSW can endure up to 6% drift without significant damage, surpassing expected ductility levels of a special moment frame, particularly with efficient slit geometry.

Effects of annealing temperature on microstructure and mechanical properties of a low density δ-TRIP steel plate via intercritical quenching-annealing process

In this study, the effects of annealing temperature on microstructure and mechanical properties of Fe–9Mn–5Al-0.3C (wt.%) low density steel plates via intercritical quenching-annealing process were investigated through field-emission scanning electron microscopy, electron backscatter diffraction, X-ray diffraction, and tensile tests. The results showed that the experimental steel maintained micro-laminated microstructure after intercritical quenching-annealing treatment, consisting of δ-ferrite band and tempered martensite lamellar. After annealing at the range of 600–800 °C following quenching of 1050 °C, tempered martensite lamellar mainly changed, and affected the mechanical properties of the steel. The annealed experimental steels exhibited wide yield strength grades (460–620 MPa) with excellent ductility (40–63 %) and low yield-tensile ratio (0.65–0.75). The proportion of austenite, the density of geometrically necessary dislocations, and high-angle grain boundary density of tempered martensite region, as well as the presence or absence of carbides, were considered to contributed to the mechanical properties. Austenitic mechanical ability was analyzed. Various annealed temperature led to the different levels of austenitic stability through changing the enrichment of alloyed elements in austenite, further causing different degrees of TRIP effect. TWIP effect existed during deformation according to estimation of SFE. The steel annealed at 740 °C had maximum uniform elongation (62.4 %) and the highest product of strength and elongation (45.2 GPa%), depending on the maximum proportion of austenite transformed into martensite during deformation. However, the 680 °C annealed steel with the highest austenitic stability exhibited deficient TRIP effect, leading to the occurrence of necking. Moreover, work hardening behavior and fracture morphology of the experimental steel were analyzed.

Abstract LB412: BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability

Abstract Nasopharyngeal Carcinoma (NPC) is a distinct type of Epstein-Barr Virus (EBV)-associated head &amp; neck cancer. It is most prevalent in Southeast Asia with a high incidence rate of ~15/100,000. While mutations of the MAPK pathways are found in nearly one-fifth of head &amp; neck squamous cell carcinoma (HNSCC) in Western countries (TCGA-HNSCC Firehose cohort, N=512), and some of which are druggable in HNSCC setting (e.g. MAPK1 and HRAS mutations), we sought to determine the cumulative mutational rate of this potentially druggable pathway in 164 micro-dissected NPC cases using recently published next-generation sequencing data (98 whole-exome and 63 whole-genome-sequenced cases). An overall rate of 9.32% cases of NPC harbored MAPK pathway mutations (16 mutations in 15 tumors). Four genes are recurrently mutated: 4 NRAS (all Q61R/L hotspot mutations), 3 HRAS (G12S, Q61R, S106L), 2 BRAF (R726H, Y647*), and 2 SHC4 (Q519R, A552T) mutations. Among which, both patients bearing BRAF mutations died of NPC, suggestive of the biological importance of BRAF mutations in NPC progression. Prompted by an independent clinical report on an advanced NPC patient with somatic co-mutations of BRAFp.G469E and KRASp.G12D being sensitive to the B-raf/C-raf inhibitor sorafenib, we first examined the biological functions and potential druggability of this particular BRAFp.G469E mutation in NPC, as it represents a hotspot mutation site across 20 cancer types (BRAFp.G469E/A/R/V/* found in 137/70655 pan-cancer samples, cBioPortal). Based on the NPC clinical report, we hypothesized that this BRAFp.G469E hotspot mutation could drive NPC tumorigenesis and might be potentially sensitive to the B-raf inhibitor sorafenib. By ectopic expression of BRAFp.G469E mutation, BRAF-wildtype (wt) and EGFP-control gene into a recently developed Epstein-Barr virus-positive nasopharyngeal carcinoma [EBV(+)NPC] cell line, NPC43, we determined the biological functions of this mutation in NPC. We showed that NPC43-BRAFp.G469E-expressing cells significantly drove a 4-fold increase in 3D tumoroid growth vs. BRAF-wt and EGFP-control cells on day 7 (flat-bottom low attachment plates). Further, wound healing assay demonstrated a significantly higher migration rate of NPC43-BRAFp.G469E cells achieving ~80% wound healing (at 96h) vs. 30% and 10% healing in BRAF-wt and EGFP-control cells. Lastly, our preliminary drug study showed that NPC43-BRAFp.G469E cells displayed higher sensitivity to low dose sorafenib (33.3nM and 100nM) than BRAF-wt and EGFP-control cells, achieving ~20% and 30% growth inhibition at 72h, vs. no growth inhibition in control cells. Thus, BRAFp.G469E hotspot mutation can drive NPC tumorigenesis, and may confer sensitivity to raf inhibitors such as sorafenib in NPC. Potential druggability of other BRAF mutations in NPC should be investigated. Acknowledgements: The study was supported by Research Impact Fund (R4015-19F), Research Grants Council, University Grants Committee, Hong Kong SAR during 6/29/20-9/7/21. Citation Format: Chun Ho Law, Zoey Wing Yee Liu, Franky Leung Chan, Tin Yu Samuel Law, Vivian W.Y. Lui. BRAFp.G469E hotspot mutation promotes nasopharyngeal carcinoma tumorigenesis with potential druggability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB412.

Abstract 4248: Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development

Abstract Introduction: Approximately 30% of advanced bladder cancer (BCa) tumors exhibit MTAP gene deletion, which has been associated with luminal subtype BCa, poor response to chemotherapy, and shorter progression to metastatic disease (De Souza 2023). MTAP deletion has been proposed as a potential biomarker for response to pemetrexed and an emerging class of PRMT5 and MAT2A inhibitors in development for MTAP-deleted tumors. Here, we describe the first patient-derived 3D model of BCa with MTAP deletion and its translational potential as a drug sensitivity platform. Methods: A patient (pt) with metastatic BCa harboring MTAP deletion underwent a pelvic lymph node biopsy. A fresh tumor sample was processed under collagenase/dispase solution for 30 minutes and filtered through a 70-um cell strainer. Cells were cultures in low attachment plates with MammoCult™ Basal Medium (Stem Cell Technologies). After 5 passages and 3D spheroid formation, immunostaining for cytokeratin (CK) 5 and 6, GATA3, CK20, and Ki67 staining were performed to confirm the urothelial origin. The MTAP-del was confirmed by a western blot of the organoid specimen and compared to other BLCa cell lines (UMUC, RT4, J82, and 5637). The viability assays were performed using the CellTiter-Glo® 3D Cell kit. Next-generation sequencing (NGS) of primary BCa tumor revealed deletion of MTAP, CDKN2A, CDKN2B, and mutations in p53, TERT, and KDM6A. Results: The patient organoid sample was positive for the luminal CK5/6 markers and negative for GATA3. Ki67 and CK20 were primarily expressed in the nucleus of the same sample. The organoid retained morphologic and immunohistochemical features of the patient’s primary bladder tumor, showing the same squamous differentiation and similar Ki-67 proliferative index. The organoid displayed no evidence of the MTAP protein by western blot (similar findings with the MTAP-del cell lines UMUC and RT4). The organoid showed sensitivity to pemetrexed [pemetrexed IC50=0.12 uM (other MTAP-del BLCa IC50: 0.1-0.23 uM)] and resistance to gemcitabine and cisplatin [Gem IC50=55 nM (other BLCa IC50: 1-29 nM), Cis IC50=90.5 uM (other BLCa IC50: 2-17 uM). Conclusions: We describe the first patient-derived MTAP-deleted BCa organoid recapitulating the features of the primary tumor. The organoid displayed sensitivity to pemetrexed as described with MTAP-deleted BCa. The organoid represents a unique platform for testing novel therapeutic strategies for MTAP-deleted BCa. Citation Format: Maximilian Schwermann, Benedito Carneiro, Shaolei Lu, Andre De Souza, Matthew Hadfield, Anthony Mega, Galina Lagos, Sari Khaleel, Dragan Golijanin, Sheldon Holder, Praveen Srinivasan, William MacDonald, Andrew George, Lanlan Zhou, Leiqing Zhang, Wafik El-Deiry. Patient-derived MTAP-deleted bladder cancer organoid model: A unique platform for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4248.

Abstract 2060: Automation of 3D cancer spheroid assay for compound screening

Abstract Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications. Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2060.

Abstract 512: Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles

Abstract Background: Traditional cell culture methods involve growing cells on flat surfaces, which do not fully replicate the human physiological conditions. In contrast, 3D cell cultures accurately reproduce aspects of the in-vitro malignant and non-malignant cell behavior. However, differences in tumor sensitivity platforms between 2D vs. 3D models might be relevant for some, but not for other drugs. Methods: The colorectal cancer cell line HCT-116 was cultured and plated in flat bottom well plates for the 2D condition and in Ultra Low Attachment round-bottom plates for the 3D condition. After plating, cells were treated with chemotherapies and targeted therapies for four days; cellular sensitivity was assessed through a viability assay. Four-parameter logistic (4PL) dose response curves were generated and IC50s were quantified to understand the differences in responses between 2D and 3D cellular environments. Results: HCT-116 exhibited similar responses to platinum drugs, taxanes, and several other tested drugs in both platforms, with the exception of gemcitabine (2D IC50 0.04 mcM; 3D IC50 12.8 mcM), lapatinib (2D IC50 23; 3D IC50 418), tucatinib (2D IC50 23.3; 3D IC50 79.3), and palbociclib (2D IC50 7.0; 3D IC50 24.0). Interestingly, these drugs share a common mechanism of action, which is the inhibition of cell cycle progression from G1 to S phase. Hence, we hypothesize that this shared mechanism may account for the observed differences in drug efficacy between the two environments. Conclusions: Similar dose responses have been observed with various drugs on both platforms, yet drugs that inhibit cell cycle progression from G1 to S phase, revealed higher sensitivity in 2D and more resistance in the 3D platform. Confirmatory cell-cycle results analyses are in progress. Further research is needed to understand if this applies to patient samples. Citation Format: Jahanvi Kumar, Chiara Maestri, Rajeshwar Nitiyanandan, Ivan Trus, Ricardo J. Parker, Brigitte Apfel, Christian Apfel. Cell-cycle inhibition may influence differences in 2D vs 3D tumor sensitivity profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 512.

Abstract 234: Microfluidic chip for drug response studies in non-small cell lung carcinoma patient-derived organoids

Abstract Background. Organoids have been widely accepted as 3D tumor models for patient-specific drug response. However, research involving non-small cell lung cancer (NSCLC) organoids is hampered by the lack of appropriate platforms. Although dome cultures show promising results, the relatively large size variability of patient-derived organoids (PDOs) hinders their application in drug response studies. Here, we report a microfluidic chip for maintaining high uniformity and requiring miniscule sample volume for PDO drug screening. Methods. The microfluidic chip includes a bottom microwell layer and a top microchannel layer. Each chip has 5 channels for parallel treatment conditions, with 30 U-shaped microwells (400μm dia x 250μm deep) per channel. PDOs were loaded from side channels and trapped by filters adjacent to microwells. NSCLC PDOs F231/F671 (NCI PDMR) were cultured and treated by KRASG12C inhibitor adagrasib (0-2000nM, 72h). PDO drug response was assessed via viability using fluorescent dyes. Results. On-chip PDO size after one-week incubation exhibited the coefficient of variation (CV) of 28% for F231 and 36% for F671, which was notably lower than measurements obtained using Matrigel dome (126%, n=3) and low attachment plate (80%, n=4). The F231/F671 showed viability &amp;gt;81% after 1wk (n=20). On-chip cytotoxicity results showed that both KRASG12C PDOs exhibited sensitivity to adagrasib, matching the clinical response, with IC50 values of 830 nM (F231) and 1324nM (F671). When cultured with fibroblast (WI-38) supernatant, the cytotoxic effect was mitigated due to the fibroblast-induced resistance, with IC50 increasing ≥2 ×. No statistical difference was observed for the fibroblast effect in the open-format microwells and our microfluidic chip (69.6±16.9% to 80.2±9.0% vs. 69.0±12.2% to 82.2±4.1%, respectively, following 500nM adagrasib for 72h), further underscoring the reliability of our chip for PDOs’ drug response studies. Application of continuous perfusion at 70nL/min yielded no statistical difference in viability from static conditions. Conclusions. Our microfluidic chip offers uniform PDO growth with perfusion allowing drug screening in almost real time. The on-chip response to adagrasib was clinically relevant. Resistance due to tumor microenvironment (TME) can be readily assessed using fibroblast supernatant, and could be expanded to other TME components in future studies to investigate their impact on treatment resistance. Citation Format: Qiyue Luan, Ines Pulido, Angelique Isagirre, Jian Zhou, Takeshi Shimamura, Ian Papautsky. Microfluidic chip for drug response studies in non-small cell lung carcinoma patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 234.

INVBSTIGATION OF SINGLE FILLET WELD FOR BUILT-UPMEMBERS

The main purpose of this study is to investigated the single fillet weld design for built-up membersby using ftision welding and find the effects of welding power input on that design for member. Inthis work, investigation of welding metal for Low carbon steel plate type St.52-3, thickness 6-8rnm, which is to ensure good mechanical properties under high stresses for ( Welding metal , heataffected Zone (HAZ) and base metal ). Also it can be explain, that the effects of welding processeson the deformation and changes in mechanical and metallurgical properties due to high thermalenergy, results from welding process. This study involved: weld design, selection of suitableelectrode, evaluation of mechanical and metallurgical properties, the maximum allowable value ofsweep of welded mamber after ( fabrication and erection ) has been calculated, also leg and throughlength have been calculated for the welded Zone. The main results of this study are :- The value of sweep which occur before the erection and tightening is ( 29 mm. ).- The leg and through length of welding metal which welded by electrode type( E 7018 = OKUS ), its found equal to ( 4 mm. ).The study results can ensure good mechanical and metallurgical properties for signal filled weld( Built-up members ).' i

Theory of nonlocal modal hydrodynamic functions for beam and plate vibrations in viscous fluids

In this paper, we introduce a new nonlocal modal hydrodynamic theory for fluid–structure interactions (FSI) of light, flexible cantilever beams and plates undergoing small amplitude vibrations in Newtonian, incompressible, viscous, heavy fluids otherwise at rest. For low aspect ratio flexible structures and high mode numbers, three dimensional (3D) and nonlocal fluid effects become prominent drivers of the coupled dynamics, to the point that existing local hydrodynamic theories based on two dimensional (2D) fluid approximations become inadequate to predict the system response. On the other hand, our approach is based on a rigorous, yet efficient, 3D treatment of the hydrodynamic loading on cantilevered thin structures. The off-line solution of the FSI problem results in the so-called nonlocal modal hydrodynamic function matrix, that is, the representation of the nonlocal hydrodynamic load operator on a basis formed by the structural modes. Our theory then integrates the nonlocal hydrodynamics within a fully coupled structural modal model in the frequency domain. We compare and discuss our theory predictions in terms of frequency response functions, mode shapes, hydrodynamic loads, quality factors, added mass ratios with the predictions of the classical local approaches, for different actuation scenarios, identifying the limitations of the hypotheses underlying existing treatments. Importantly, we also validate our new model with experiments conducted on flexible square plates. While computationally efficient, our fully coupled theory is exact up to numerical truncation and can bridge knowledge gaps in the design and analysis of FSI systems based on low aspect ratio flexible beams and plates.