Targets Of Interest Research Articles

Abstract 12726: Novel, Potent, and Highly Selective Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Inhibitors Reduce Substrate Phosphorylation in Rat Hearts and Prolong Survival in a Mouse Model of Severe Heart Failure

Introduction: CaMKII is a target of longstanding interest in drug development and has been validated in multiple preclinical models of heart failure, as well as in arrhythmia diseases, including catecholaminergic polymorphic ventricular tachycardia and atrial fibrillation. We describe here the pharmacological profiles of a novel oral CaMKII inhibitor, CRD-2015. We demonstrate the ability of CRD-2015 to reduce substrate phosphorylation in rat hearts, and to strongly prolong survival in the calsequestrin transgenic (CSQ-Tg) mouse model of severe heart failure. Results: CRD-2015 is a potent (IC 50 0.62nM), highly selective CaMKII inhibitor. Oral administration of CRD-2015 dose-dependently decreased phospholamban (P-PLN) and ryanodine receptor 2 (P-RyR2) phosphorylation at their CaMKII-specific sites (Thr17 and Ser2814, respectively) in normal rat hearts, with strong correlation between the plasma concentrations (PK) and the reduction in phosphorylation (PD) (Figure, A). In CSQ-Tg mice, CRD-2015 also decreased cardiac P-PLN and P-RyR2 in a dose-dependent manner. Chronic administration of CRD-2015 prolonged survival in a dose-dependent manner (Figure, B). At the maximum dose level, all CSQ-Tg mice survived for 27 days (n=15/15) with the treatment of CRD-2015, while no mice survived past 24 days in the vehicle-treated group (n=0/15). Similar dose and concentration-dependent reductions in P-PLN and P-RyR2 and prolongation of survival in CSQ-Tg mice were also detected with a second potent (IC 50 0.40nM) and selective CaMKII inhibitor, CRD-2959. Conclusions: Our novel CaMKII inhibitor, CRD-2015 shows a clear PK/PD relationship in normal rats and prolongs survival in a mouse model of severe heart failure. These results demonstrate the successful creation of specific CaMKII inhibitors with efficacy in preclinical models and support their clinical development for the treatment of heart failure and other cardiovascular diseases.

Differential specificity of SARS-CoV-2 main protease variants on peptide versus protein-based substrates.

The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.

Sequentially-Activated Antibodies Based on Programmable DNA Tags for Rapid Multiplexed Protein Imaging.

The achievement of rapid multiplexed protein imaging is limited by the use of stimulating reagents, extensive incubating and washing steps, and the low fluorescence intensity of targets. In this study, sequentially-activated DNA tags are developed and combined them with primary antibodies using signal enhancement strategies to create sequentially-activated antibodies (SAAs). These SAAs enable rapid, wash-free sequential imaging of different protein targets. The samples are pre-processed to label all targets of interest with SAAs simultaneously, and the signal is turned ON for only one target in each stage. The sequential imaging of multiple targets is achieved through wash-free strand displacement reactions that exhibit rapid kinetics with t1/2 <10s in a cellular context. Remarkably, this method successfully demonstrates sequential imaging of nine different protein targets within just a few minutes. This all-in-one platform for multiplexed protein imaging holds great promise for diverse applications in immunofluorescence imaging.

Generation of a helper phage for the fluorescent detection of peptide-target interactions by dual-display phages

Phage display is a molecular biology technique that allows the presentation of foreign peptides on the surface of bacteriophages. It is widely utilized for applications such as the discovery of biomarkers, the development of therapeutic antibodies, and the investigation of protein–protein interactions. When employing phages in diagnostic and therapeutic monitoring assays, it is essential to couple them with a detection system capable of revealing and quantifying the interaction between the peptide displayed on the phage capsid and the target of interest. This process is often technically challenging and costly. Here, we generated a fluorescent helper phage vector displaying sfGFP in-frame to the pIII of the capsid proteins. Further, we developed an exchangeable dual-display phage system by combining our newly developed fluorescent helper phage vector with a phagemid vector harboring the engineered pVIII with a peptide-probe. By doing so, the sfGFP and a peptide-probe are displayed on the same phage particle. Notably, our dual-display approach is highly flexible as it allows for easy exchange of the displayed peptide-probe on the pVIII to gain the desired selectivity, while maintaining the sfGFP gene, which allows easy visualization and quantification of the interaction peptide-probe. We anticipate that this system will reduce time and costs compared to the current phage-based detection systems.

Multisensor Agile Adaptive Sampling (MAAS): A Methodology to Collect Radar Observations of Convective Cell Life Cycle

Abstract Multisensor Agile Adaptive Sampling (MAAS), a smart sensing framework, was adapted to increase the likelihood of observing the vertical structure (with little to no gaps), spatial variability (at subkilometer scale), and temporal evolution (at ∼2-min resolution) of convective cells. This adaptation of MAAS guided two mechanically scanning C-band radars (CSAPR2 and CHIVO) by automatically analyzing the latest NEXRAD data to identify, characterize, track, and nowcast the location of all convective cells forming in the Houston domain. MAAS used either a list of predetermined rules or real-time user input to select a convective cell to be tracked and sampled by the C-band radars. The CSAPR2 tracking radar was first tasked to collect three sector plan position indicator (PPI) scans toward the selected cell. Edge computer processing of the PPI scans was used to identify additional targets within the selected cell. In less than 2 min, both the CSAPR2 and CHIVO radars were able to collect bundles of three to six range–height indicator (RHI) scans toward different targets of interest within the selected cell. Bundles were successively collected along the path of cell advection for as long as the cell met a predetermined set of criteria. Between 1 June and 30 September 2022 over 315 000 vertical cross-section observations were collected by the C-band radars through ∼1300 unique isolated convective cells, most of which were observed for over 15 min of their life cycle. To the best of our knowledge, this dataset, collected primarily through automatic means, constitutes the largest dataset of its kind.

Automated Detection and Tracking of Marine Mammals in the Vicinity of Tidal Turbines Using Multibeam Sonar

Understanding how marine animals behave around tidal turbines is essential if we are to quantify how individuals and populations may be affected by the installation of these devices in the coming decades. Our particular interest is in collision risk, and how this may be affected by the fine-scale behaviour of seals and small cetacean species around devices. We report on a study in which multibeam sonar data were collected close to an operational tidal turbine in Scotland continuously over a twelve-month period. The sonars provide high-resolution (a few cm) data over a 120° angle out to a range of 55 m at a rate of 10 frames per second. We describe a system which uses automatic computer algorithms to detect potential targets of interest, verified by human analysts using a sophisticated computer user interface to confirm detections and assign target species. To date, we have identified 359 tracks of marine mammals in the data, as well as several thousand tracks from fish and diving birds. These are currently being parameterised to study how these species react to the moving turbine rotors, and the data are now being used to explore the development of improved automated detection and classification algorithms.

Clutter Rejection for FM Ranging Airborne Radar

In airborne radar, it is important to reject clutter signals to detect targets of interest. High pulse repetition frequency (HPRF) waveform provides robust detection performance in a clutter environment by separating clutter signals from the target echo signals in a Doppler frequency domain, but it suffers from range ambiguity. The range ambiguity in HPRF can be resolved by frequency modulation (FM) ranging. However, the Doppler frequencies of both the clutter and target echo signals change linearly with the range due to changes in the carrier frequency in FM ranging. In such a case, the target echo signal can be rejected by a conventional clutter rejection frequency even if it is not masked by a clutter signal. This paper proposes an optimum clutter rejection frequency for FM ranging airborne radar by considering the Doppler frequency spread in FM ranging. The optimum clutter rejection frequency is derived by calculating the maximum Doppler frequency of the spread clutter signal caused by FM ranging. The simulation and flight test results verify that the proposed clutter rejection frequency is the optimum value that can improve the detection capability of FM ranging airborne radar.

Targeted Therapies in Advanced Cholangiocarcinoma.

Primary tumor resection and liver transplantation are the only curative treatment options for the management of cholangiocarcinoma (CCA). However, for patients with advanced or metastatic disease, palliative systemic therapy remains the only treatment option. The development of targeted therapeutics has begun to shift the treatment paradigm in CCA. Targets of interest in CCA include mutated isocitrate dehydrogenase-1 (mIDH-1), human epidermal growth factor receptor 2 (HER2) overexpression/amplification, and fibroblast growth factor receptor 2 (FGFR2) fusion, in addition to less frequently observed targets such as BRAF V600E, deficient mismatch repair/high microsatellite instability (dMMR/MSI-H), and high tumor mutation burden (TMB-H). These targets are observed in varying frequency among patients with intrahepatic CCA and extrahepatic CCA. Multiple novel therapies have been developed to exploit each of these targets, with some having received United States Food and Drug Administration approval for use in the second-line setting. In the current review, we discuss targets of interest in CCA and summarize current evidence evaluating available therapies directed at these targets.

Selenium doped carbonized polypyrrole with high peroxidase-like activity for determining D-penicillamine

Via one-step pyrolysis of polypyrrole and Se powder mixture at a low temperature (450 °C), we reported the fabrication of Se doped carbonized polypyrrole (Se-CP) as nanozyme for determining D-penicillamine (D-PA). After in situ selenium doping, more defect sites and large specific surface area were endowed to Se-CP. Selenium doping in carbon skeleton generated unique C-Se-C bonds, which were conducive to enhancing the asymmetric spin and charge density around the carbon atom, resulting in the acceleration of electron transfer and high catalytic activity. The Se-CP exhibited the capacity to rapidly catalyze the oxidation of TMB by H2O2, and its peroxidase-like activity has increased by 6.51-fold compared to carbonized polypyrrole (CP). The Se-CP exhibited high affinity for substrates TMB and H2O2 with the Km values of 0.0820 mM and 0.182 mM, respectively. Based on the inhibition of D-PA on the high catalytic activity of Se-CP, a sensitive and convenient colorimetric sensing platform has been established for its detection with a detection limit of 60.0 nM. This method was successfully applied to detect the content of penicillamine in actual pharmaceuticals. This work provides a simple and mild strategy to prepare high performance nanozyme for detecting targets of interest.

Not-so-fast Kepler-1513: a perturbing planetary interloper in the exomoon corridor

ABSTRACT Transit timing variations (TTVs) can be induced by a range of physical phenomena, including planet–planet interactions, planet–moon interactions, and stellar activity. Recent work has shown that roughly half of moons would induce fast TTVs with a short period in the range of 2–4 orbits of its host planet around the star. An investigation of the Kepler TTV data in this period range identified one primary target of interest, Kepler-1513 b. Kepler-1513 b is a $8.05^{+0.58}_{-0.40}$ R⊕ planet orbiting a late G-type dwarf at $0.53^{+0.04}_{-0.03}$ au. Using Kepler photometry, this initial analysis showed that Kepler-1513 b’s TTVs were consistent with a moon. Here, we report photometric observations of two additional transits nearly a decade after the last Kepler transit using both ground-based observations and space-based photometry with TESS. These new transit observations introduce a previously undetected long period TTV, in addition to the original short period TTV signal. Using the complete transit data set, we investigate whether a non-transiting planet, a moon, or stellar activity could induce the observed TTVs. We find that only a non-transiting perturbing planet can reproduce the observed TTVs. We additionally perform transit origami on the Kepler photometry, which independently applies pressure against a moon hypothesis. Specifically, we find that Kepler-1513 b’s TTVs are consistent with an exterior non-transiting ∼Saturn mass planet, Kepler-1513 c, on a wide orbit, $\sim 5~{{\ \rm per \, cent}}$ outside a 5:1 period ratio with Kepler-1513 b. This example introduces a previously unidentified cause for planetary interlopers in the exomoon corridor, namely an insufficient baseline of observations.

Extract2Chip-Bypassing Protein Purification in Drug Discovery Using Surface Plasmon Resonance.

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein-protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein-protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply.

FRI290 Proteomic Identification Of Secretoneurin-Stimulated Proteins In Purified Mouse Gonadotropes

Abstract Disclosure: A. Herdman: None. A. Lagasse: None. A. Haney: None. S. Byrum: None. U. Boehm: None. M.C. MacNicol: None. A.M. MacNicol: None. G.V. Childs: None. A.K. Odle: None. Secretoneurin (SN) is a proteolytic product of secretogranin II (SCG2), a member of the granin family of proteins. Both SCG2 and SN have been localized in the anterior pituitary (AP), and SCG2 co-localizes to gonadotrope granules containing luteinizing hormone (LH). Preliminary studies from others have shown that exogenous SN is capable of stimulating LH secretion from the mouse LßT2 cell line and goldfish pituitaries, and SN may provide a paracrine signal from other AP cells to support LH production/secretion. Despite the implications for regulatory reproductive signaling, a comprehensive study of the effect of SN on female mammalian gonadotropes has not been conducted. To define the pathways and processes affected by exogenous secretoneurin (SN) on murine gonadotrope protein expression, pituitaries from 18 proestrous female mice expressing Gnrhr-driven Cre and a floxed fluorescent Cre reporter were collected and dispersed. Half of the dispersed pituitaries were stimulated with 1 μM SN, and all cells were incubated for 3 hours. Gonadotropes were purified via fluorescence-activated cell sorting. After pooling to optimize cell counts, control and SN-stimulated gonadotropes were submitted for mass-spectrometry proteomics. A differential expression analysis was performed to determine fold-change (FC) in proteins from SN-stimulated samples over control (unstimulated), and false discovery rate (FDR)-controlled adjusted p values were used to assign significance (Adj p≤0.05). A total of 948 proteins were identified in the purified gonadotrope pools. Of these, 136 proteins were significantly differentially expressed between control and SN-stimulated gonadotrope pools; 53 proteins were upregulated and 83 were downregulated in SN-stimulated gonadotropes. Upregulated targets of particular interest include the SN precursor SCG2 (FC = 12, p&amp;lt;0.05), as well as prohormone convertase 2 (PC2: FC = 8, p&amp;lt;0.05) which can cleave SCG2 to produce SN. Also upregulated is ELAV-like protein 1 (ELAVL1: FC=5, p&amp;lt;0.05), an RNA-binding protein known to regulate gonadotropin-releasing hormone receptor (GnRHR) expression and bind other gonadotrope-critical mRNAs. Although LHß was unchanged, the glycoprotein alpha subunit (CGA) was decreased 4-fold following SN stimulation (p = 0.05). This is consistent with previous reports showing decreased Cga mRNA following SN stimulation. In addition to the expected “cell signaling” and “response to stimulation”, gene ontology analysis showed that the major biological processes altered by SN stimulation include processing of RNA (including RNA binding, translation regulation, etc.) and regulation of cell structure. Indeed, several RNA-binding proteins and actin-related proteins were significantly changed. Our data confirm previous results from LßT2 cells and define new SN targets and pathways that may play a broader role in gonadotrope physiology. Presentation: Friday, June 16, 2023

Space-time cluster analysis of anemia in pregnant women in the province of Khyber Pakhtunkhwa, Pakistan (2014-2020).

Anaemia is a common public-health problem affecting about two-thirds of pregnant women in developing countries. Spacetime cluster analysis of anemia cases is important for publichealth policymakers to design evidence-based intervention strategies. This study discovered the potential space-time clusters of anemia in pregnant women in Khyber Pakhtunkhwa Province, Pakistan, from 2014 to 2020 using space-time scan statistic (SatScan). The results show that the most likely cluster of anemia was seen in the rural areas in the eastern part of the province covering five districts from 2017 to 2019. However, three secondary clusters in the West and one in the North were still active, signifying important targets of interest for public-health interventions. The potential anemia clusters in the province's rural areas might be associated with the lack of nutritional education in women and lack of access to sufficient diet due to financial constraints.

MRT-ModSeq – Rapid Detection of RNA Modifications with MarathonRT

Chemical modifications are essential regulatory elements that modulate the behavior and function of cellular RNAs. Despite recent advances in sequencing-based RNA modification mapping, methods combining accuracy and speed are still lacking. Here, we introduce MRT-ModSeq for rapid, simultaneous detection of multiple RNA modifications using MarathonRT. MRT-ModSeq employs distinct divalent cofactors to generate 2-D mutational profiles that are highly dependent on nucleotide identity and modification type. As a proof of concept, we use the MRT fingerprints of well-studied rRNAs to implement a general workflow for detecting RNA modifications. MRT-ModSeq rapidly detects positions of diverse modifications across a RNA transcript, enabling assignment of m1acp3Y, m1A, m3U, m7G and 2′-OMe locations through mutation-rate filtering and machine learning. m1A sites in sparsely modified targets, such as MALAT1 and PRUNE1 could also be detected. MRT-ModSeq can be trained on natural and synthetic transcripts to expedite detection of diverse RNA modification subtypes across targets of interest.

Integrated procedures for accelerating, deepening, and leading genetic inquiry: A first application on human muscle secretome

PurposeBeyond classical procedures, bioinformatic-assisted approaches and computational biology offer unprecedented opportunities for scholars. However, these amazing possibilities still need epistemological criticism, as well as standardized procedures. Especially those topics with a huge body of data may benefit from data science (DS)-assisted methods. Therefore, the current study dealt with the combined expert-assisted and DS-assisted approaches to address the broad field of muscle secretome. We aimed to apply DS tools to fix the literature research, suggest investigation targets with a data-driven approach, predict possible scenarios, and define a workflow. MethodsRecognized scholars with expertise on myokines were invited to provide a list of the most important myokines. GeneRecommender, GeneMANIA, HumanNet, and STRING were selected as DS tools. Networks were built on STRING and GeneMANIA. The outcomes of DS tools included the top 5 recommendations. Each expert-led discussion has been then integrated with an DS-led approach to provide further perspectives. ResultsAmong the results, 11 molecules had already been described as bona-fide myokines in literature, and 11 molecules were putative myokines. Most of the myokines and the putative myokines recommended by the DS tools were described as present in the cargo of extracellular vesicles. ConclusionsIncluding both supervised and unsupervised learning methods, as well as encompassing algorithms focused on both protein interaction and gene represent a comprehensive approach to tackle complex biomedical topics. DS-assisted methods for reviewing existent evidence, recommending targets of interest, and predicting original scenarios are worth exploring as in silico recommendations to be integrated with experts' ideas for optimizing molecular studies.

Serotonin: A new super effective functional monomer for molecular imprinting. The case of TNF-[formula omitted] detection in real matrix by Surface Plasmon Resonance

Molecular imprinting and related technologies are becoming increasingly appreciated in bioanalysis and diagnostic applications. Among the imprinted polymers, we have already demonstrated that the endogenous neurotransmitters (NTs) dopamine (DA) and norepinephrine (NE) can be efficiently used as natural and sustainable monomers to straightforwardly design and synthesize a new generation of green and “soft” Molecularly Imprinted BioPolymers (MIBPs). Here, we demonstrated for the first time the ability of a further NT, i.e., serotonin (SE), in forming adhesive imprinted nanofilms coupled to label-free optical biosensing. Its imprinting efficiency is compared with those obtained with PDA and PNE. As a model study, tumor necrosis factor-alpha (TNF-α) was selected as a biomolecular target of interest in clinical diagnostics. The biomimetic receptor was coupled to Surface Plasmon Resonance (SPR), and TNF-α detection was performed in label-free and real-time manner both in buffer and biological matrices, i.e. synovial fluid and human serum. The results indicate that, under the same imprinting and binding conditions, the analytical performances of PSE are impressively superior to those of PDA and PNE. The PSE-based MIBP was able to detect TNF-α in human matrices with a good sensitivity, selectivity, and repeatability.

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.

Identification of GDC-1971 (RLY-1971), a SHP2 Inhibitor Designed for the Treatment of Solid Tumors.

Protein tyrosine phosphatase SHP2 mediates RAS-driven MAPK signaling and has emerged in recent years as a target of interest in oncology, both for treating with a single agent and in combination with a KRAS inhibitor. We were drawn to the pharmacological potential of SHP2 inhibition, especially following the initial observation that drug-like compounds could bind an allosteric site and enforce a closed, inactive state of the enzyme. Here, we describe the identification and characterization of GDC-1971 (formerly RLY-1971), a SHP2 inhibitor currently in clinical trials in combination with KRAS G12C inhibitor divarasib (GDC-6036) for the treatment of solid tumors driven by a KRAS G12C mutation.

Real-Time Precision Needle Tracking for Gynecological Interstitial Brachytherapy: A Phantom Feasibility Study

To determine if an advanced needle navigation and real-time precision tracking system developed for prostate biopsies can be used for the purpose of real-time guidance of interstitial needles in modern image-guided gynecologic brachytherapy. An agarose gel pelvic phantom was previously designed using 3D Slicer and Autodesk Fusion360 to model an average uterus from a 50-patient dataset, a vaginal canal from KleenSpec (TM, Welch Allyn) speculum dimensions, and a rectum to accommodate a BK 8848 endocavity biplane ultrasound [US] probe. After manufacturing the phantom with a 7.2cm x 3.7cm x 4.4cm (LxWxH) uterus and an attached 2.7cm x 1.4cmx 1.7cm (LxWxH) focal HR-CTV target, the model was imaged using MRI. The uterus and HR-CTV were contoured on MRI to aid in registration with US and provide a target for needle placement. The tracking system with endorectal probe was then used to image the phantom. Ultrasound images from a 180-degree sweep were acquired and a 3D Ultrasound-based volume was generated. Using the commercially available system, the MRI was imported and an MRI-Ultrasound fusion performed based on the uterus contours and then refined using contour-based deformable registration. Three desired locations for needle placement were defined to test the ability of the system to guide needles at these locations: one in the most lateral aspect of the HRCTV, one at the HRCTV-uterus interface, and one inside the uterus. The needle tracking sleeve that was indexed to the probe was used in conjunction with the software interface to guide the 17-gauge titanium needle placement in real time. The distance between actual needle and the intended needle location was measured for each successful needle placement. When possible, needles were removed and reinserted at the same desired location to test the reproducibility of insertion under US guidance. Two of the three intended needles were successfully placed using real-time US guidance. The most lateral needle in the HRCTV was not inserted due to limitations of the phantom design, specifically inaccessibility to the agarose phantom through the phantom wall supports. The inserted needles were visible and tracked to the desired location within 0.6 mm (needle located at HRCTV-uterus interface), and 2.2 mm (needles inserted centrally in the uterus). For the target within the uterus, needles were reinserted at the same target of interest with a ∆ 0.15mm in greatest range between attempts. We have demonstrated a proof of concept in which existing technology for prostate biopsies can be utilized for the application of interstitial gynecologic brachytherapy with a high degree of fidelity. Ongoing modifications to our validation phantom and the tracking system are needed to allow for variations in uterine anatomy, more rigorous evaluation of reproducibility, and multi-catheter placement.

A Novel Planning and Delivery Technology: Dose, Dose Rate and Linear Energy Transfer (LET) Optimization Based on Spot-Scanning Proton Arc Therapy FLASH (SPLASHLET)

To achieve a high conformal dose with Linear Energy Transfer (LET) optimized FLASH proton therapy, we introduced a new planning and delivery technique concept, the voxel-wised optimization of LET distribution and dose rate based on scanning arc therapy (SPLASHLET) MATERIALS/METHODS: The algorithm optimizes (1) the clinical dose-volume constraint based on dose distribution and (2) the clinical LET-volume constraint based on LET distribution using Alternating Direction Method of Multipliers (ADMM) with Limited-memory BFGS solver by minimizing the monitor unit (MU) constraint on spot weight and (3) the effective dose-average dose rate by minimizing the accelerator's beam current sequentially. Such optimization framework enables the high dose conformal dynamic arc therapy with the capability of LET painting with voxel-based FLASH dose rate in an open-source proton planning platform (MatRad, Department of Medical Physics in Radiation Oncology, German Cancer Research Center-DKFZ). It aiming to minimize the overall cost function value combined with plan quality and voxel-based LET and dose rate constraints. Three representative cases (brain, liver and prostate cancer) were used for testing purposes. Dose-volume histogram (DVH), LET volume histogram (LVH) dose rate volume histogram (DRVH) and dose rate map were assessed compared to the original SPArc plan (SPArcoriginal). SPLASHLET plan could offer comparable plan quality compared to SPArcoriginal plan. The DRVH results indicated that SPArcoriginal could not achieve FLASH using the clinic beam current configuration, while SPLASHLET could significantly not only improve V40Gy/s in target and region of interest (ROI) but also improve the mean LET in the target and reduce the high LET in organ at risk (OAR) in comparison with SPArcoriginal (Table 1). SPLASHLET offers the first LET painting with voxel-based ultra-dose-rate and high-dose conformity treatment using proton beam therapy. Such technique has the potential to take full vantage of LET painting, FLASH and SPArc.