Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Deployable ultrasound applicators for endoluminal delivery of volumetric hyperthermia.

PURPOSE: To investigate the design of an endoluminal deployable ultrasound applicator for delivering volumetric hyperthermia to deep tissue sites as a possible adjunct to radiation and chemotherapy. METHOD: This study considers an ultrasound applicator consisting of two tubular transducers situated at the end of a catheter assembly, encased within a distensible conical shaped balloon-based reflector that redirects acoustic energy distally into the tissue. The applicator assembly can be inserted endoluminally or laparoscopically in a compact form and expanded after delivery to the target site. Comprehensive acoustic and biothermal simulations and parametric studies were employed in generalized 3D and patient-specific pancreatic head and body tumor models to characterize the acoustic performance and evaluate heating capabilities of the applicator by investigating the device at a range of operating frequencies, tissue acoustic and thermal properties, transducer configurations, power modulation, applicator positioning, and by analyzing the resultant 40, 41, and 43 °C isothermal volumes and penetration depth of the heating volume. Intensity distributions and volumetric temperature contours were calculated to define moderate hyperthermia boundaries. RESULTS: Parametric studies demonstrated the frequency selection to control volume and depth of therapeutic heating from 62 to 22 cm3 and 4 to 2.6 cm as frequency ranges from 1 MHz to 4.7 MHz, respectively. Width of the heating profile tracks closely with the aperture. Water cooling within the reflector balloon was effective in controlling temperature to 37 °C maximum within the luminal wall. Patient-specific studies indicated that applicators with extended OD in the range of 3.6-6.2 cm with 0.5-1 cm long and 1 cm OD transducers can heat volumes of 1.1-7 cm3, 3-26 cm3, and 3.3-37.4 cm3 of pancreatic body and head tumors above 43, 41, and 40 °C, respectively. CONCLUSION: In silico studies demonstrated the feasibility of combining endoluminal ultrasound with an integrated expandable balloon reflector for delivering volumetric hyperthermia in regions adjacent to body lumens and cavities.

Assessing high-intensity focused ultrasound treatment of prostate cancer with hyperpolarized 13 C dual-agent imaging of metabolism and perfusion.

The goal of the study was to establish early hyperpolarized (HP) 13 C MRI metabolic and perfusion changes that predict effective high-intensity focused ultrasound (HIFU) ablation and lead to improved adjuvant treatment of partially treated regions. To accomplish this a combined HP dual-agent (13 C pyruvate and 13 C urea) 13 C MRI/multiparametric 1 H MRI approach was used to measure prostate cancer metabolism and perfusion 3-4 h, 1 d, and 5 d after exposure to ablative and sub-lethal doses of HIFU within adenocarcinoma of mouse prostate tumors using a focused ultrasound applicator designed for murine studies. Pathologic and immunohistochemical analysis of the ablated tumor demonstrated fragmented, non-viable cells and vasculature consistent with coagulative necrosis, and a mixture of destroyed tissue and highly proliferative, poorly differentiated tumor cells in tumor tissues exposed to sub-lethal heat doses in the ablative margin. In ablated regions, the intensity of HP 13 C lactate or HP 13 C urea and dynamic contrast-enhanced (DCE) MRI area under the curve images were reduced to the level of background noise by 3-4 h after treatment with no recovery by the 5 d time point in either case. In the tissues that received sub-lethal heat dose, there was a significant 60% ± 12.4% drop in HP 13 C lactate production and a significant 30 ± 13.7% drop in urea perfusion 3-4 h after treatment, followed by recovery to baseline by 5 d after treatment. DCE MRI Ktrans showed a similar trend to HP 13 C urea, demonstrating a complete loss of perfusion with no recovery in the ablated region, while having a 40%-50% decrease 3-4 h after treatment followed by recovery to baseline values by 5 d in the margin region. The utility of the HP 13 C MR measures of perfusion and metabolism in optimizing focal HIFU, either alone or in combination with adjuvant therapy, deserves further testing in future studies.

Endobronchial high-intensity ultrasound for thermal therapy of pulmonary malignancies: simulations with patient-specific lung models.

Objective: This study investigates the feasibility of endobronchial ultrasound applicators for thermal ablation of lung tumors using acoustic and biothermal simulations.Methods: Endobronchial ultrasound applicators with planar (10 mm width) or tubular transducers (6 mm outer diameter (OD)) encapsulated by expandable coupling balloons (10 mm OD) are considered for treating tumors from within major airways; smaller catheter-based applicators with tubular transducers (1.7-4 mm OD) and coupling balloons (2.5-5 mm OD) are considered within deep lung airways. Parametric studies were applied to evaluate transducer configurations, tumor size and location, effects of acoustic reflection and absorption at tumor-lung parenchyma interfaces, and the utility of lung flooding for enhancing accessibility. Patient-specific anatomical lung models, with various geometries and locations of tumors, were developed for further evaluation of device performance and treatment strategies. Temperature and thermal dose distributions were calculated and reported.Results: Large endobronchial applicators with planar or tubular transducers (3-7 MHz, 5 min) can thermally ablate tumors attached to major bronchi at up to 3 cm depth, where reflection and attenuation of normal lung localize tumor heating; with lung flooding, endobronchial applicators can ablate ∼2 cm diameter tumors with up to ∼2 cm separation from the bronchial wall, without significant heating of intervening tissue. Smaller catheter-based tubular applicators can ablate tumors up to 2-3 cm in diameter from deep lung airways (5-9 MHz, 5 min).Conclusion: Simulations demonstrate the feasibility of endobronchial ultrasound applicators to deliver thermal coagulation of 2-3 cm diameter tumors adjacent to or accessible from major and deep lung airways.

Transurethral high-intensity ultrasound for treatment of stress urinary incontinence (SUI): simulation studies with patient-specific models.

BACKGROUND: Stress urinary incontinence (SUI) is prevalent in adult women, attributed to weakened endopelvic supporting tissues, and typically treated using drugs and invasive surgical procedures. The objective of this in silico study is to explore transurethral high-intensity ultrasound for delivery of precise thermal therapy to the endopelvic tissues adjacent to the mid-urethra, to induce thermal remodeling as a potential minimally invasive treatment alternative. METHODS: 3D acoustic (Rayleigh-Sommerfeld) and biothermal (Pennes bioheat) models of the ultrasound applicator and surrounding tissues were devised. Parametric studies over transducer configuration [frequency, radius-of-curvature (ROC)] and treatment settings (power, duration) were performed, and select cases on patient-specific models were used for further evaluation. Transient temperature and thermal dose distributions were calculated, and temperature and dose metrics reported. RESULTS: Configurations using a 5-MHz curvilinear transducer (3.5 × 10 mm, 28 mm ROC) with single 90 s sonication can create heated zones with 11 mm penetration (>50 °C) while sparing the inner 1.8 mm (<45 °C) radial depth of the urethral mucosa. Sequential and discrete applicator rotations can sweep out bilateral coagulation volumes (1.4 W power, 15° rotations, 600 s total time), produce large volumetric (1124 mm³ above 60 EM43 °C) and wide angular (∼50.5° per lateral sweep) coverage, with up to 15.6 mm thermal penetration and at least 1.6 mm radial urethral protection (<5 EM43 °C). CONCLUSION: Transurethral applicators with curvilinear ultrasound transducers can deliver spatially selective temperature elevations to lateral mid-urethral targets as a possible means to tighten the endopelvic fascia and adjacent tissues.

Thermal therapy of pancreatic tumours using endoluminal ultrasound: Parametric and patient-specific modelling.

PURPOSE: The aim of this study is to investigate endoluminal ultrasound applicator configurations for volumetric thermal ablation and hyperthermia of pancreatic tumours using 3D acoustic and biothermal finite element models. MATERIALS AND METHODS: Parametric studies compared endoluminal heating performance for varying applicator transducer configurations (planar, curvilinear-focused, or radial-diverging), frequencies (1-5 MHz), and anatomical conditions. Patient-specific pancreatic head and body tumour models were used to evaluate feasibility of generating hyperthermia and thermal ablation using an applicator positioned in the duodenal or stomach lumen. Temperature and thermal dose were calculated to define ablation (> 240 EM(43 °C)) and moderate hyperthermia (40-45 °C) boundaries, and to assess sparing of sensitive tissues. Proportional-integral control was incorporated to regulate maximum temperature to 70-80 °C for ablation and 45 °C for hyperthermia in target regions. RESULTS: Parametric studies indicated that 1-3 MHz planar transducers are the most suitable for volumetric ablation, producing 5-8 cm(3) lesion volumes for a stationary 5-min sonication. Curvilinear-focused geometries produce more localised ablation to 20-45 mm depth from the GI tract and enhance thermal sparing (T(max) < 42 °C) of the luminal wall. Patient anatomy simulations show feasibility in ablating 60.1-92.9% of head/body tumour volumes (4.3-37.2 cm(3)) with dose < 15 EM(43 °C) in the luminal wall for 18-48 min treatment durations, using 1-3 applicator placements in GI lumen. For hyperthermia, planar and radial-diverging transducers could maintain up to 8 cm(3) and 15 cm(3) of tissue, respectively, between 40-45 °C for a single applicator placement. CONCLUSIONS: Modelling studies indicate the feasibility of endoluminal ultrasound for volumetric thermal ablation or hyperthermia treatment of pancreatic tumour tissue.

Coherence between cellular responses and in vitro splicing inhibition for the anti-tumor drug pladienolide B and its analogs.

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.

An endoluminal cylindrical sectored-ring ultrasound phased-array applicator for minimally-invasive therapeutic ultrasound.

BACKGROUND: The size of catheter-based ultrasound devices for delivering ultrasound energy to deep-seated tumors is constrained by the access pathway which limits their therapeutic capabilities. PURPOSE: To devise and investigate a deployable applicator suitable for minimally-invasive delivery of therapeutic ultrasound, consisting of a 2D cylindrical sectored-ring ultrasound phased array, integrated within an expandable paraboloid-shaped balloon-based reflector. The balloon can be collapsed for compact delivery and expanded close to the target position to mimic a larger-diameter concentric-ring sector-vortex array for enhanced dynamic control of focal depth and volume. METHODS: Acoustic and biothermal simulations were employed in 3D generalized homogeneous and patient-specific heterogeneous models, for three-phased array transducers with 32, 64, and 128 elements, composed of sectored 4, 8, and 16 tubular ring transducers, respectively. The applicator performance was characterized as a function of array configuration, focal depth, phasing modes, and balloon reflector geometry. A 16-element proof-of-concept phased array applicator assembly, consisting of four tubular transducers each divided into four sectors, was fabricated, and characterized with hydrophone measurements along and across the axis, and ablations in ex vivo tissue. RESULTS: Simulation results indicated that transducer arrays (1.5 MHz, 9 mm OD × 20 mm long), balloon sizes (41-50 mm expanded diameter, 20-60 mm focal depth), phasing mode (0-4) and sonication duration (30 s) can produce spatially localized acoustic intensity focal patterns (focal length: 3-22 mm, focal width: 0.7-8.7 mm) and ablative thermal lesions (width: 2.7-16 mm, length: 6-46 mm) in pancreatic tissue across a 10-90 mm focal depth range. Patient-specific studies indicated that 0.1, 0.46, and 1.2 cm3 volume of tumor can be ablated in the body of the pancreas for 120 s sonications using a single axial focus (Mode 0), or four, and eight simultaneous foci in a toroidal pattern (Mode 2 and 4, respectively). Hydrophone measurements demonstrated good agreement with simulation. Experiments in which chicken meat was thermally ablated indicated that volumetric ablation can be produced using single or multiple foci. CONCLUSIONS: The results of this study demonstrated the feasibility of a novel compact ultrasound applicator design capable of focusing, deep penetration, electronic steering, and volumetric thermal ablation. The proposed applicator can be used for compact endoluminal or laparoscopic delivery of localized ultrasound energy to deep-seated targets.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

High Contrast Ultrasonic Method With Multi-Spatiotemporal Compounding for Monitoring Catheter-Based Ultrasound Thermal Therapy: Development and Ex Vivo Evaluations.

OBJECTIVE: Changes in ultrasound backscatter energy (CBE) imaging can monitor thermal therapy. Catheter-based ultrasound (CBUS) can treat deep tumors with precise spatial control of energy deposition and ablation zones, of which CBE estimation can be limited by low contrast and robustness due to small or inconsistent changes in ultrasound data. This study develops a multi-spatiotemporal compounding CBE (MST-CBE) imaging approach for monitoring specific to CBUS thermal therapy. METHODS: Ex vivo thermal ablations were performed with stereotactic positioning of a 180° directional CBUS applicator, temperature monitoring probes, endorectal US probe, and subsequent lesion sectioning and measurement. Five frames of raw radiofrequency data were acquired throughout in 15s intervals. Using window-by-window estimation methods, absolute and positive components of MST-CBE images at each point were obtained by the compounding ratio of squared envelope data within an increasing spatial size in each short-time window. RESULTS: Compared with conventional US, Nakagami, and CBE imaging, the detection contrast and robustness quantified by tissue-modification-ratio improved by 37.2 ± 4.7 (p < 0.001), 37.5 ± 5.2 (p < 0.001), and 6.4 ± 4.0 dB (p < 0.05) in the MST-CBE imaging, respectively. Correlation coefficient and bias between cross-sectional dimensions of the ablation zones measured in tissue sections and estimated from MST-CBE were up to 0.91 (p < 0.001) and -0.02 mm2, respectively. CONCLUSION: The MST-CBE approach can monitor the detailed changes within target tissues and effectively characterize the dimensions of the ablation zone during CBUS energy deposition. SIGNIFICANCE: The MST-CBE approach could be practical for improved accuracy and contrast of monitoring and evaluation for CBUS thermal therapy.

Generation of recombinant hyperimmune globulins from diverse B-cell repertoires.

Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.

In silico feasibility assessment of extracorporeal delivery of low-intensity pulsed ultrasound to intervertebral discs within the lumbar spine.

Low intensity pulsed ultrasound (LIPUS) may have utility for non-invasive treatment of discogenic lower back pain through stimulating, remodeling and accelerating healing of injured or degenerated intervertebral disc (IVD) tissues. This study investigates the feasibility of delivering LIPUS to lumbar IVDs between L2 and S1 spine vertebra using a planar extracorporeal phased array (8 × 8 cm, 1024 elements, 500 kHz). Three 3D anatomical models with heterogenous tissues were generated from patient CT image sets and used in the simulation-based analysis. Time-reversal acoustic modeling techniques were applied to optimize posterior-lateral placement of the array with respect to the body to facilitate energy deposition in discrete target regions spanning the annulus fibrosus and central nucleus of each IVD. Forward acoustic and biothermal simulations were performed with time-reversal optimized array placements and driving amplitude/phase settings to predict LIPUS intensity distributions at target sites and to investigate off-target energy deposition and heating potential. Simulation results demonstrate focal intensity gain of 5-168 across all IVD targets and anatomical models, with greater average intensity gain (>50) and energy localization in posterior, posterolateral, and lateral target sites of IVDs. Localized LIPUS delivery was enhanced in thinner patient anatomies and in the high lumbar levels (L2-L3 and L3-L4). Multiple amplitude/phasing illumination patterns could be sequenced at a fixed array position for larger regional energy coverage in the IVD. Biothermal simulations demonstrated that LIPUS-appropriate exposures of 100 mW cm-2 ISPTA to the target disc region would result in <1 °C global peak temperature elevation for all cases. Hence, simulations suggest that spatially-precise extracorporeal delivery of therapeutically relevant LIPUS doses to discrete regions of lumbar IVDs is feasible and may be useful in clinical management of discogenic back pain.

LIPUS far-field exposimetry system for uniform stimulation of tissues in-vitro: development and validation with bovine intervertebral disc cells.

Therapeutic Low-intensity Pulsed Ultrasound (LIPUS) has been applied clinically for bone fracture healing and has been shown to stimulate extracellular matrix (ECM) metabolism in numerous soft tissues including intervertebral disc (IVD). In-vitro LIPUS testing systems have been developed and typically include polystyrene cell culture plates (CCP) placed directly on top of the ultrasound transducer in the acoustic near-field (NF). This configuration introduces several undesirable acoustic artifacts, making the establishment of dose-response relationships difficult, and is not relevant for targeting deep tissues such as the IVD, which may require far-field (FF) exposure from low frequency sources. The objective of this study was to design and validate an in-vitro LIPUS system for stimulating ECM synthesis in IVD-cells while mimicking attributes of a deep delivery system by delivering uniform, FF acoustic energy while minimizing reflections and standing waves within target wells, and unwanted temperature elevation within target samples. Acoustic field simulations and hydrophone measurements demonstrated that by directing LIPUS energy at 0.5, 1.0, or 1.5 MHz operating frequency, with an acoustic standoff in the FF (125-350 mm), at 6-well CCP targets including an alginate ring spacer, uniform intensity distributions can be delivered. A custom FF LIPUS system was fabricated and demonstrated reduced acoustic intensity field heterogeneity within CCP-wells by up to 93% compared to common NF configurations. When bovine IVD cells were exposed to LIPUS (1.5 MHz, 200 µs pulse, 1 kHz pulse frequency, and ISPTA = 120 mW cm-2) using the FF system, sample heating was minimal (+0.81 °C) and collagen content was increased by 2.6-fold compared to the control and was equivalent to BMP-7 growth factor treatment. The results of this study demonstrate that FF LIPUS exposure increases collagen content in IVD cells and suggest that LIPUS is a potential noninvasive therapeutic for stimulating repair of tissues deep within the body such as the IVD.

Deployable cylindrical phased-array applicator mimicking a concentric-ring configuration for minimally-invasive delivery of therapeutic ultrasound.

A novel design for a deployable catheter-based ultrasound applicator for endoluminal and laparoscopic intervention is introduced. By combining a 1D cylindrical ring phased array with an expandable paraboloid or conical-shaped balloon-based reflector, the applicator can be controllably collapsed for compact delivery and deployed to mimic a forward-firing larger diameter concentric ring array with tight focusing and electronic steering capabilities in depth. Comprehensive acoustic and biothermal parametric studies were employed to characterize the capabilities of the applicator design as a function of transducer dimensions, phased array configuration, and balloon reflector geometry. Modeling results indicate that practical balloon sizes (43-57 mm expanded diameter), transducer array configurations (e.g. 1.5 MHz, 10 mm OD × 20 mm length, 8 or 16 array elements), and sonication durations (30 s) are capable of producing spatially-localized acoustic intensity focal patterns and ablative thermal lesions (width: 2.8-4.8 mm; length: 5.3-40.1 mm) in generalized soft tissue across a 5-100 mm depth range. Larger focal intensity gain magnitudes and narrower focal dimensions are attainable using paraboloid-shaped balloon reflectors with natural geometric focal depths of 25-55 mm, whereas conical-shaped reflectors (angled 45-55°) produce broader foci and extend electronic steering range in depth. A proof-of-concept phased array applicator assembly was fabricated and characterized using hydrophone and radiation force balance measurements and demonstrated good agreement with simulation. The results of this study suggest that combining small diameter cylindrical phased arrays with expandable balloon reflectors can enhance minimally invasive ultrasound-based intervention by augmenting achievable focal gains and penetration depths with dynamic adjustment of treatment depth.

Noninvasive, Targeted Creation of Neuromyelitis Optica Pathology in AQP4-IgG Seropositive Rats by Pulsed Focused Ultrasound.

Neuromyelitis optica spectrum disorders (herein called NMO) is an autoimmune disease of the CNS characterized by astrocyte injury, inflammation, and demyelination. In seropositive NMO, immunoglobulin G autoantibodies against aquaporin-4 (AQP4-IgG) cause primary astrocyte injury. A passive transfer model of NMO was developed in which spatially targeted access of AQP4-IgG into the CNS of seropositive rats was accomplished by pulsed focused ultrasound through intact skin. Following intravenous administration of microbubbles, pulsed ultrasound at 0.5 MPa peak acoustic pressure was applied using a 1 MHz transducer with 6-cm focal length. In brain, the transient opening of the blood-brain barrier (BBB) in an approximately prolate ellipsoidal volume of diameter ∼3.5 mm and length ∼44 mm allowed entry of IgG-size molecules for up to 3-6 hours. The ultrasound treatment did not cause erythrocyte extravasation or inflammation. Ultrasound treatment in AQP4-IgG seropositive rats produced localized NMO pathology in brain, with characteristic astrocyte injury, inflammation, and demyelination after 5 days. Pathology was not seen when complement was inhibited, when non-NMO human IgG was administered instead of AQP4-IgG, or in AQP4-IgG seropositive AQP4 knockout rats. NMO pathology was similarly created in cervical spinal cord in seropositive rats. These results establish a noninvasive, spatially targeted model of NMO in rats, and demonstrate that BBB permeabilization, without underlying injury or inflammation, is sufficient to create NMO pathology in AQP4-IgG seropositive rats.

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics.

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

Theoretical investigation of transgastric and intraductal approaches for ultrasound-based thermal therapy of the pancreas.

BACKGROUND: The goal of this study was to theoretically investigate the feasibility of intraductal and transgastric approaches to ultrasound-based thermal therapy of pancreatic tumors, and to evaluate possible treatment strategies. METHODS: This study considered ultrasound applicators with 1.2 mm outer diameter tubular transducers, which are inserted into the tissue to be treated by an endoscopic approach, either via insertion through the gastric wall (transgastric) or within the pancreatic duct lumen (intraductal). 8 patient-specific, 3D, transient, biothermal and acoustic finite element models were generated to model hyperthermia (n = 2) and ablation (n = 6), using sectored (210°-270°, n = 4) and 360° (n = 4) transducers for treatment of 3.3-17.0 cm3 tumors in the head (n = 5), body (n = 2), and tail (n = 1) of the pancreas. A parametric study was performed to determine appropriate treatment parameters as a function of tissue attenuation, blood perfusion rates, and distance to sensitive anatomy. RESULTS: Parametric studies indicated that pancreatic tumors up to 2.5 or 2.7 cm diameter can be ablated within 10 min with the transgastric and intraductal approaches, respectively. Patient-specific simulations demonstrated that 67.1-83.3% of the volumes of four sample 3.3-11.4 cm3 tumors could be ablated within 3-10 min using transgastric or intraductal approaches. 55.3-60.0% of the volume of a large 17.0 cm3 tumor could be ablated using multiple applicator positions within 20-30 min with either transgastric or intraductal approaches. 89.9-94.7% of the volume of two 4.4-11.4 cm3 tumors could be treated with intraductal hyperthermia. Sectored applicators are effective in directing acoustic output away from and preserving sensitive structures. When acoustic energy is directed towards sensitive structures, applicators should be placed at least 13.9-14.8 mm from major vessels like the aorta, 9.4-12.0 mm from other vessels, depending on the vessel size and flow rate, and 14 mm from the duodenum. CONCLUSIONS: This study demonstrated the feasibility of generating shaped or conformal ablative or hyperthermic temperature distributions within pancreatic tumors using transgastric or intraductal ultrasound.

Integration of deployable fluid lenses and reflectors with endoluminal therapeutic ultrasound applicators: Preliminary investigations of enhanced penetration depth and focal gain.

PURPOSE: Catheter-based ultrasound applicators can generate thermal ablation of tissues adjacent to body lumens, but have limited focusing and penetration capabilities due to the small profile of integrated transducers required for the applicator to traverse anatomical passages. This study investigates a design for an endoluminal or laparoscopic ultrasound applicator with deployable acoustic reflector and fluid lens components, which can be expanded after device delivery to increase the effective acoustic aperture and allow for deeper and dynamically adjustable target depths. Acoustic and biothermal theoretical studies, along with benchtop proof-of-concept measurements, were performed to investigate the proposed design. METHODS: The design schema consists of an array of tubular transducer(s) situated at the end of a catheter assembly, surrounded by an expandable water-filled conical balloon with a secondary reflective compartment that redirects acoustic energy distally through a plano-convex fluid lens. By controlling the lens fluid volume, the convex surface can be altered to adjust the focal length or collapsed for device insertion or removal. Acoustic output of the expanded applicator assembly was modeled using the rectangular radiator method and secondary sources, accounting for reflection and refraction at interfaces. Parametric studies of transducer radius (1-5 mm), height (3-25 mm), frequency (1.5-3 MHz), expanded balloon diameter (10-50 mm), lens focal length (10-100 mm), lens fluid (silicone oil, perfluorocarbon), and tissue attenuation (0-10 Np/m/MHz) on beam distributions and focal gain were performed. A proof-of-concept applicator assembly was fabricated and characterized using hydrophone-based intensity profile measurements. Biothermal simulations of endoluminal ablation in liver and pancreatic tissue were performed for target depths between 2 and 10 cm. RESULTS: Simulations indicate that focal gain and penetration depth scale with the expanded reflector-lens balloon diameter, with greater achievable performance using perfluorocarbon lens fluid. Simulations of a 50 mm balloon OD, 10 mm transducer outer diameter (OD), 1.5 MHz assembly in water resulted in maximum intensity gain of ~170 (focal dimensions: ~12 mm length × 1.4 mm width) at ~5 cm focal depth and focal gains above 100 between 24 and 84 mm depths. A smaller (10 mm balloon OD, 4 mm transducer OD, 1.5 MHz) configuration produced a maximum gain of 6 at 9 mm depth. Compared to a conventional applicator with a fixed spherically focused transducer of 12 mm diameter, focal gain was enhanced at depths beyond 20 mm for assembly configurations with balloon diameters ≥ 20 mm. Hydrophone characterizations of the experimental assembly (31 mm reflector/lens diameter, 4.75 mm transducer radius, 1.7 MHz) illustrated focusing at variable depths between 10-70 mm with a maximum gain of ~60 and demonstrated agreement with theoretical simulations. Biothermal simulations (30 s sonication, 75 °C maximum) indicate that investigated applicator assembly configurations, at 30 mm and 50 mm balloon diameters, could create localized ellipsoidal thermal lesions increasing in size from 10 to 55 mm length × 3-6 mm width in liver tissue as target depth increased from 2 to 10 cm. CONCLUSIONS: Preliminary theoretical and experimental analysis demonstrates that combining endoluminal ultrasound with an expandable acoustic reflector and fluid lens assembly can significantly enhance acoustic focal gain and penetration from inherently smaller diameter catheter-based applicators.

Rare, high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade, discovered using microfluidics and molecular genomics.

Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with KD < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.