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Purpose: Despite recent approvals for checkpoint inhibitors and antibody–drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain–containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. Experimental Design: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). Results: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. Conclusions: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

Congenital diarrheas and enteropathies (CODEs) constitute a heterogeneous group of individually rare disorders manifesting with infantile-onset chronic diarrhea. Genomic deletions in chromosome 16, encompassing a sequence termed the ‘intestine-critical region (ICR)’, were recently identified as the cause of an autosomal recessive congenital enteropathy. The regulatory sequence within the ICR is flanked by an unannotated open reading frame termed PERCC1, which plays a role in enteroendocrine cell (EEC) function. We investigated two unrelated children with idiopathic congenital diarrhea requiring home parenteral nutrition attending the Irish Intestinal Failure Program. Currently 12 and 19-years old, these Irish male patients presented with watery diarrhea and hypernatremic dehydration in infancy. Probands were phenotyped by comprehensive clinical investigations, including endoscopic biopsies and serum gastrin level measurements. Following negative exome sequencing, PCR and Sanger sequencing of the entire coding region and intron boundaries of PERCC1 were performed for each proband and their parents. In both patients, serum gastrin levels were low and failed to increase following a meal challenge. While no deletions involving the ICR were detected, targeted sequencing of the PERCC1 gene revealed a shared homozygous c.390C > G stop gain variant. We report clinical and molecular findings in two unrelated patients harboring a shared homozygous variant in PERCC1, comprising the first description of a point mutation in this gene in association with CODE. That both parenteral nutrition dependent children with unexplained diarrhea at our institution harbored a PERCC1 mutation underscores the importance of its inclusion in exome sequencing interpretation.

We employed a previously described procedure, based on circular dichroism (CD) spectroscopy, to quantify the distribution of conformational states adopted by equimolar mixtures of complementary G-rich and C-rich DNA strands from the promoter regions of the VEGF and Bcl-2 oncogenes. Spectra were recorded at different pHs, concentrations of KCl, and temperatures. The temperature dependences of the fractional populations of the duplex, G-quadruplex

The advent of autonomous electric vehicles (AEVs) will give drivers time and space instead of focusing on driving. Because of this, some drivers may want to personalize their car for their work, while others may want to customize their vehicle space to be more suitable for relaxation, which will accelerate the megatrend of mass individualization. However, the production of individualized cars faces several challenges. For example, since high-level automation during individualized car production is difficult, a stable skilled labor supply is essential, low-volume/high-variety production is required, and customer proximity or involvement is also important. These conditions can be satisfied by building a car assembly plant in an urban area. The problem is that urban areas are often spatially and environmentally constrained. However, it is be possible to overcome these urban limitations by implementing a conveyor-less micro factory. The objective of this study is to propose a new iterative matrix-system layout design method that can realize a conveyor-less urban car assembly factory with two technologies—VaaC (vehicle as a conveyor) and matrix assembly system. VaaC consists of three novel ideas: sensor skid, safety-sensor guidance system, and vehicle-powered devices, and this paper views each of them in detail. The proposed iterative matrix-system layout design method consists of four steps: (1) layout refinement, (2) simulation, (3) cost analysis, and (4) optimization check, and will examine how each step is performed through simple examples. The authors hope that this paper will arouse interest and provide elements to spur future research on the conveyor-less urban car assembly system.

A major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

SABER (Sounding of the Atmosphere using Broadband Emission Radiometry) is a 10-channel infrared radiometer that is one of four instruments on the NASA TIMED (Thermosphere-Ionosphere-Mesosphere Energetics and Dynamics) satellite mission to study the structure, energetics, chemistry, and dynamics of the Earth’s mesosphere and lower thermosphere. The TIMED spacecraft was launched into a 625 km circular polar orbit (74.1º inclination) via a Boeing Delta II rocket from Vandenberg Air Force Base on 7 December 2001. SABER continues to operate nominally and collect data routinely as it has for over 21 years. Over 2,200 peer-reviewed journal articles have been published worldwide using SABER data. A list of these articles is included in the Supporting Information accompanying this paper. This paper presents a detailed technical description of the SABER instrument including major subsystems of the instrument and technical performance parameters. This paper comprehensively describes the instrument and its components and provides final instrument design and performance parameters. The motivation for this paper is to document this information permanently for future reference. The Space Dynamics Laboratory (SDL) of Utah State University designed, fabricated, and calibrated the SABER instrument in close collaboration with NASA Langley Research Center, Hampton University, and Global Atmospheric Technologies and Science (GATS).

Barrier-to-Autointegration Factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1’s DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1’s N-terminal domain.

In this paper, a conflict detection system for small Unmanned Aerial Vehicles (sUAS), composed of an interacting multiple model state predictor and a Haversine-distance based conflict detector, is proposed. The conflict detection system was developed and tested via a random recursive simulation in the ROS-Gazebo physics engine environment. The simulation consisted of ten small unmanned aerial vehicles flying along randomly assigned way-point navigation missions within a confined airspace. Way-points are generated from a uniform distribution and then sent to each vehicle. The interacting multiple model state predictor runs on a ground-based system and only has access to current vehicle positional information. It does not have access to the future way-points of individual vehicles. The state predictor is based on Kalman filters that utilize constant velocity, constant acceleration, and constant turn models. It generates near-future position estimates for all vehicles operating within an airspace. These models are probabilistically fused together and projected into the near-future to generate state predictions. These state predictions are then passed to the Haversine distance-based conflict detection algorithm to compare state estimates and identify probable conflicts. The conflicts are detected and flagged based on tunable threshold values which compare distances between predictions for the vehicles operating within the airspace. This paper discusses the development of the random recursive simulation for the ROS-Gazebo framework and the derivation of the interacting multiple model along-with the Haversine-based future conflict detector. The results are presented via simulation to highlight mid-air conflict detection application for sUAS operations in the National Airspace.

The in vitro differentiation of pluripotent stem cells into human intestinal organoids (HIOs) has served as a powerful means for creating complex three-dimensional intestinal structures. Owing to their diverse cell populations, transplantation into an animal host is supported with this system and allows the temporal formation of fully laminated structures, including crypt-villus architecture and smooth muscle layers that resemble native human intestine. Although the endpoint of HIO engraftment has been well described, here we aim to elucidate the developmental stages of HIO engraftment and establish whether it parallels fetal human intestinal development. We analyzed a time course of transplanted HIOs histologically at 2, 4, 6 and 8 weeks post-transplantation, and demonstrated that HIO maturation closely resembles key stages of fetal human intestinal development. We also utilized single-nuclear RNA sequencing to determine and track the emergence of distinct cell populations over time, and validated our transcriptomic data through in situ protein expression. These observations suggest that transplanted HIOs do indeed recapitulate early intestinal development, solidifying their value as a human intestinal model system.

Human leukocyte antigens (HLA) display peptides largely from intracellular proteins on the surface of cells in major histocompatibility complex (MHC)-peptide complexes. These complexes provide a biological window into the cell, and peptides derived from disease-associated antigens can serve as biomarkers and therapeutic targets. Thus, proper identification of peptides and the corresponding presenting HLA allele in disease phenotypes is important for the design and execution of therapeutic strategies using engineered T-cell receptors or antibodies. Yet, current mass spectrometry methods for profiling the immunopeptidome typically require large and complex sample inputs, complicating the study of several disease phenotypes and lowering the confidence of both peptide and allele identification. Here, we describe a novel secreted HLA (sHLA) Fc-fusion construct that allows for simple peptide identification from single HLA alleles in two important disease models: hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. We identify hypoxia and senescence-associated peptides that could act as future targets for immunotherapy. More generally, the method streamlines the time between sample preparation and injection from days to hours, yielding allele-restricted target identification in a temporally controlled manner. Overall, this method identified >30,000 unique HLA-associated peptides across two different HLA alleles and seven cell lines. Notably, ∼9,300 of these unique HLA-associated peptides had previously not been identified in the Immune Epitope Database. We believe the sHLA Fc-fusion capture technology will accelerate the study of the immunopeptidome as therapeutic interest in HLA-peptide complexes increases in cancer and beyond.

Purpose:Despite recent approvals for checkpoint inhibitors and antibody–drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain–containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies.Experimental Design:CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376).Results:CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively.Conclusions:These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

CDCP1 mRNA is not correlated NECTIN4 or TROP2 mRNA, related to Figure 2C. Spearman correlation analyses were performed between CDCP1 and NECTIN4 or TROP2 for the Seiler, Sjodahl 2017, and Sjodahl 2012 datasets, with p values and correlation coefficients shown. Points are colored by consensus subtype as in Figure 1.

CDCP1 mRNA is overexpressed in basal/squamous subtypes of bladder cancer, related to Figure 1A-D. Pairwise comparisons of CDCP1 expression between bladder cancer consensus subtypes using the Wilcoxon rank-sum test across four different datasets are shown.

Targeted protein degradation is a promising therapeutic strategy capable of overcoming the limitations of traditional occupancy-based inhibitors. By ablating all of the associated functions of a protein at once, the event-driven pharmacology of degrader technologies has recently enabled the targeting of proteins that have been historically deemed "undruggable". Most degradation strategies utilize the ubiquitin-proteasome system to mediate intracellular target degradation and are thus limited to targeting proteins with cytoplasmic domains. While some of these strategies, such as PROTACs, have shown great promise, there is a need for new modalities that can be applied to specifically target cell surface proteins. We previously described the development of an antibody-based PROTAC (AbTAC) that utilizes genetically encoded IgG bispecific antibody scaffolds to bring the cell surface E3-ligase RNF43 into the proximity of a membrane protein of interest (POI) to mediate its degradation. Here, we employ rational protein engineering strategies to interrogate and optimize the properties necessary for efficient degradation of two therapeutically important membrane proteins, PD-L1 and EGFR. We develop multiple antibodies to RNF43 and show that the specific antibody binding epitopes on RNF43 and the POI are more important than the affinities of the AbTAC antibodies. We further expand the available repertoire of E3 ligases by co-opting the E3-ligase ZNRF3 to degrade both PD-L1 and EGFR and show similar importance of epitope for degradation efficiency. Importantly, we show that both RNF43 and ZNRF3 AbTACs do not potentiate unwanted WNT signaling. Lastly, we find that these AbTACs can be even further improved by exploring various dual-binding and IgG scaffolds that range in flexibility, valency, and orientation of the binding arms. These structure-activity and mechanistic studies provide a roadmap for optimizing the development of AbTACs, thereby greatly expanding their utility for targeted cell surface protein degradation.

Background Small airways are known to be affected early in the course of chronic obstructive pulmonary disease (COPD); however, traditional spirometric indices may not accurately identify small airways disease. Research Question Can FEV3/FEV6 identify early airflow abnormalities and predict future clinically important respiratory-related outcomes, including development of COPD? Study Design and Methods We included eight hundred thirty-two current and former smokers with post-bronchodilator FEV1/FVC ≥0.7 from the SPIROMICS cohort. Participants were classified as having a reduced pre-bronchodilator FEV3/FEV6 based on lower limit of normal (LLN) values. Repeatability analysis was performed for FEV3 and FEV6. Regression modeling was used to evaluate the relationship between baseline FEV3/FEV6 and outcome measures including functional small airways disease on thoracic imaging and respiratory exacerbations. Interval censored analysis was used to assess progression to COPD. Results FEV3/FEV6 Interpretation FEV3/FEV6 is a routinely available and repeatable spirometric index which can be useful in the evaluation of early airflow obstruction in current and former smokers without COPD. A reduced FEV3/FEV6 can identify those at risk for future development of COPD and respiratory exacerbations.

Senescent cells undergo a permanent cell cycle arrest and drive a host of age-related pathologies. Recent transgenic mouse models indicate that removing cells expressing the senescence marker p16

PURPOSE OF REVIEW: The intestinal enteroendocrine cells (EECs) are specialized hormone secreting cells that respond to both circulating and luminal cues. Collectively, EECs constitute the largest endocrine organ of the body and signal to a multitude of targets including locally to neighboring intestinal cells, enteric neurons, as well as systemically to other organs such as the pancreas and brain. To accomplish their wide range of downstream signaling effects, EECs secrete multiple hormones; however, the mechanisms that influence EEC development in the embryo and differentiation in adults are not well-defined. RECENT FINDINGS: This review highlights the recent discoveries in EEC differentiation and function while also discussing newly revealed roles of transcription factors and signaling networks involved in the allocation of EEC subtypes that were discovered using a combination of novel intestinal model systems and genetic sequencing. We also discuss the potential of these new experimental models that study the mechanisms regulating EEC function and development both to uncover novel therapeutic targets. SUMMARY: Several EEC hormones are being used to treat various metabolic disorders such as type 2 diabetes and obesity. Therefore, understanding the signaling pathways and gene regulatory networks that facilitate EEC formation is paramount to the development of novel therapies.

PDF file, 59K, Supplementary Figure S1. A typical example of Bilateral Inguinal Lymph node size 8 days after vaccination with the native peptide QQNPSYDSV (#14) or its modified derivative YLNPSVDSV (#24).

Supplementary Table from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Purpose:The recent emergence of radioligand therapies for cancer treatment has increased enthusiasm for developing new theranostic strategies coupling both imaging and cytotoxicity in the same entity. In this study, we evaluated whether CUB domain containing protein 1 (CDCP1), a single-pass transmembrane protein highly overexpressed in diverse human cancers, might be a target for cancer theranostics.Experimental Design:The ectodomain of CDCP1 was targeted using radiolabeled forms of 4A06, a potent and specific recombinant human antibody that we developed. Imaging and antitumor assessment studies were performed in animal models of pancreatic cancer, including two patient-derived xenograft models we developed for this study. For antitumor assessment studies, the endpoints were death due to tumor volume >3,000 mm3 or ≥20% loss in body weight. Specific tracer binding or antitumor effects were assessed with an unpaired, two-tailed Student t test and survival advantages were assessed with a log rank (Mantel–Cox) test. Differences at the 95% confidence level were interpreted to be significant.Results:89Zr-4A06 detected a broad dynamic range of full length or cleaved CDCP1 expression on seven human pancreatic cancer tumors (n = 4/tumor). Treating mice with single or fractionated doses of 177Lu-4A06 significantly reduced pancreatic cancer tumor volume compared with mice receiving vehicle or unlabeled 4A06 (n = 8; P < 0.01). A single dose of 225Ac-4A06 also inhibited tumor growth, although the effect was less profound compared with 177Lu-4A06 (n = 8; P < 0.01). A significant survival advantage was imparted by 225Ac-4A06 (HR = 2.56; P < 0.05).Conclusions:These data establish that CDCP1 can be exploited for theranostics, a finding with widespread implications given its breadth of overexpression in cancer.

Purpose: The Ewing sarcoma family of tumors (ESFT) comprises a group of aggressive, malignant bone, and soft tissue tumors that predominantly affect children and young adults. These tumors frequently share expression of the EWS-FLI-1 translocation, which is central to tumor survival but not present in healthy cells. In this study, we examined EWS-FLI-1 antigens for their capacity to induce immunity against a range of ESFT types.Design: Computer prediction analysis of peptide binding, HLA-A2.1 stabilization assays, and induction of cytotoxic T-lymphocytes (CTL) in immunized HLA-A2.1 transgenic mice were used to assess the immunogenicity of native and modified peptides derived from the fusion region of EWS-FLI-1 type 1. CTL-killing of multiple ESFT family members in vitro, and control of established xenografts in vivo, was assessed. We also examined whether these peptides could induce human CTLs in vitro.Results: EWS-FLI-1 type 1 peptides were unable to stabilize cell surface HLA-A2.1 and induced weak CTL activity against Ewing sarcoma cells. In contrast, peptides with modified anchor residues induced potent CTL killing of Ewing sarcoma cells presenting endogenous (native) peptides. The adoptive transfer of CTL specific for the modified peptide YLNPSVDSV resulted in enhanced survival of mice with established Ewing sarcoma xenografts. YLNPSVDSV-specific CTL displayed potent killing of multiple ESFT types in vitro: Ewing sarcoma, pPNET, Askin's Tumor, and Biphenotypic sarcoma. Stimulation of human peripheral blood mononuclear cells with YLNPSVDSV peptide resulted in potent CTL-killing.Conclusions: These data show that YLNPSVDSV peptide is a promising antigen for ESFT immunotherapy and warrants further clinical development. Clin Cancer Res; 18(19); 5341–51. ©2012 AACR.

Supplementary Figure from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Purpose:With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.Experimental Design:CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post–enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.Results:CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4–2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4–2B xenografts.Conclusions:The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Supplemental methods, figures, and tables

We study the statistical significances for exclusion and discovery of proton decay at current and future neutrino detectors. Various counterintuitive flaws associated with frequentist and modified frequentist statistical measures of significance for multi-channel counting experiments are discussed in a general context and illustrated with examples. We argue in favor of conservative Bayesian-motivated statistical measures, and as an application we employ these measures to obtain the current lower limits on proton partial lifetime at various confidence levels, based on Super-Kamiokande's data, generalizing the 90\% CL published limits. Finally, we present projections for exclusion and discovery reaches for proton partial lifetimes in $p \rightarrow \overline \nu K^+$ and $p \rightarrow e^+ \pi^0$ decay channels at Hyper-Kamiokande, DUNE, JUNO, and THEIA., Comment: 58 pages. v2: published in PRD with minor revisions

Human organoids have potential to revolutionizein vitrodisease modeling by providing multicellular architecture and function that are similar to thosein vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering “microarray three-dimensional (3D) bioprinting” technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids forin situfunctional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal–immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases.