Neoantigen-specific stem cell memory-like CD4+ T cells mediate CD8+ T cell-dependent immunotherapy of MHC class II-negative solid tumors.

CD4+ T cells play key roles in a range of immune responses, either as direct effectors or through accessory cells, including CD8+ T lymphocytes. In cancer, neoantigen (NeoAg)-specific CD8+ T cells capable of direct tumor recognition have been extensively studied, whereas the role of NeoAg-specific CD4+ T cells is less well understood. We have characterized the murine CD4+ T cell response against a validated NeoAg (CLTCH129>Q) expressed by the MHC-II-deficient squamous cell carcinoma tumor model (SCC VII) at the level of single T cell receptor (TCR) clonotypes and in the setting of adoptive immunotherapy. We find that the natural CLTCH129>Q-specific repertoire is diverse and contains TCRs with distinct avidities as measured by tetramer-binding assays and CD4 dependence. Despite these differences, CD4+ T cells expressing high or moderate avidity TCRs undergo comparable in vivo proliferation to cross-presented antigen from growing tumors and drive similar levels of therapeutic immunity that is dependent on CD8+ T cells and CD40L signaling. Adoptive cellular therapy (ACT) with NeoAg-specific CD4+ T cells is most effective when TCR-engineered cells are differentiated ex vivo with IL-7 and IL-15 rather than IL-2 and this was associated with both increased expansion as well as the acquisition and stable maintenance of a T stem cell memory (TSCM)-like phenotype in tumor-draining lymph nodes (tdLNs). ACT with TSCM-like CD4+ T cells results in lower PD-1 expression by CD8+ T cells in the tumor microenvironment and an increased frequency of PD-1+CD8+ T cells in tdLNs. These findings illuminate the role of NeoAg-specific CD4+ T cells in mediating antitumor immunity via providing help to CD8+ T cells and highlight their therapeutic potential in ACT.

Screening for generality in asymmetric catalysis.

Research in the field of asymmetric catalysis over the past half century has resulted in landmark advances, enabling the efficient synthesis of chiral building blocks, pharmaceuticals and natural products1-3. A small number of asymmetric catalytic reactions have been identified that display high selectivity across a broad scope of substrates; not coincidentally, these are the reactions that have the greatest impact on how enantioenriched compounds are synthesized4-8. We postulate that substrate generality in asymmetric catalysis is rare not simply because it is intrinsically difficult to achieve, but also because of the way chiral catalysts are identified and optimized9. Typical discovery campaigns rely on a single model substrate, and thus select for high performance in a narrow region of chemical space. Here we put forth a practical approach for using multiple model substrates to select simultaneously for both enantioselectivity and generality in asymmetric catalytic reactions from the outset10,11. Multisubstrate screening is achieved by conducting high-throughput chiral analyses by supercritical fluid chromatography-mass spectrometry with pooled samples. When applied to Pictet-Spengler reactions, the multisubstrate screening approach revealed a promising and unexpected lead for the general enantioselective catalysis of this important transformation, which even displayed high enantioselectivity for substrate combinations outside of the screening set.

Allogeneic B cell immunomodulatory therapy in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an orphan neurodegenerative disease. Immune system dysregulation plays an essential role in ALS onset and progression. Our preclinical studies have shown that the administration of exogenous allogeneic B cells improves outcomes in murine models of skin and brain injury through a process termed pligodraxis, in which B cells adopt an immunoregulatory and neuroprotective phenotype in an injured environment. Here, we investigated the effects of B-cell therapy in the SOD1G93A mouse preclinical model of ALS and in a person living with ALS. Purified splenic mature naïve B cells from haploidentical donor mice were administered intravenously in SOD1G93A mice for a total of 10 weekly doses. For the clinical study in a person with advanced ALS, IgA gammopathy of unclear significance, and B lymphopenia, CD19+ B cells were positively selected from a healthy haploidentical donor and infused intravenously twice, at a 60-day interval. Repeated intravenous B-cell administration was safe and significantly delayed disease onset, extended survival, reduced cellular apoptosis, and decreased astrogliosis in SOD1G93A mice. Repeated B-cell infusion in a person with ALS was safe and did not appear to generate a clinically evident inflammatory response. An improvement of 5 points on the ALSFRS-R scale was observed after the first infusion. Levels of inflammatory markers showed persistent reduction post-infusion. This represents a first demonstration of the efficacy of haploidentical B-cell infusion in the SOD1G93A mouse and the safety and feasibility of using purified haploidentical B lymphocytes as a cell-based therapeutic strategy for a person with ALS.

Identification of gene fusions associated with amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Genetics is an important risk factor for amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. Recent findings demonstrate that in addition to specific genetic mutations, structural variants caused by genetic instability can also play a causative role in ALS. Genomic instability can lead to deletions, duplications, insertions, inversions, and translocations in the genome, and these changes can sometimes lead to fusion of distinct genes into a single transcript. Gene fusion events have been studied extensively in cancer; however, they have not been thoroughly investigated in ALS. The aim of this study was to determine whether gene fusions are present in ALS. METHODS: Gene fusions were identified using STAR Fusion v1.10.0 software in bulk RNA-Seq data from human postmortem samples from publicly available data sets from Target ALS and the New York Genome Center ALS Consortium. RESULTS: We report the presence of gene fusion events in several brain regions as well as in spinal cord samples in ALS. Although most gene fusions were intra-chromosomal events between neighboring genes and present in both ALS and control samples, there was a significantly greater number of unique gene fusions in ALS compared to controls. Lastly, we identified specific gene fusions with a significant burden in ALS, that were absent from both control samples and known cancer gene fusion databases. DISCUSSION: Collectively, our findings reveal an enrichment of gene fusions in ALS and suggest that these events may be an additional genetic cause linked to ALS pathogenesis.

Phase space analysis of two-wavelength interferometry.

Multiple wavelength phase shifting interferometry is widely used to extend the unambiguous range (UR) beyond that of a single wavelength. Towards this end, many algorithms have been developed to calculate the optical path difference (OPD) from the phase measurements of multiple wavelengths. These algorithms fail when phase error exceeds a specific threshold. In this paper, we examine this failure condition. We introduce a "phase-space" view of multi-wavelength algorithms and demonstrate how this view may be used to understand an algorithm's robustness to phase measurement error. In particular, we show that the robustness of the synthetic wavelength algorithm deteriorates near the edges of its UR. We show that the robustness of de Groot's extended range algorithm [Appl. Opt.33, 5948 (1994)APOPAI0003-693510.1364/AO.33.005948] depends on both wavelength and OPD in a non-trivial manner. Further, we demonstrate that the algorithm developed by Houairi and Cassaing (HC) [J. Opt. Soc. Am. A26, 2503 (2009)JOAOD60740-323210.1364/JOSAA.26.002503] results in uniform robustness across the entire UR. Finally, we explore the effect that wavelength error has on the robustness of the HC algorithm.

Experimental and Theoretical Exploration of the Kinetics and Thermodynamics of the Nucleophile-Induced Fragmentation of Ylidenenorbornadiene Carboxylates.

Ylidenenorbornadienes (YNDs), prepared by [4 + 2] cycloadditions between fulvenes and acetylene carboxylates, react with thiol nucleophiles to yield mixtures of four to eight diastereomers depending on the symmetry of the YND substrate. The mixtures of diastereomers fragment via a retro-[4 + 2] cycloaddition with a large variation in rate, with half-lives ranging from 16 to 11,000 min at 80 °C. The diastereomer-enriched samples of propane thiol adducts [YND-propanethiol (PTs)] were isolated and identified by nuclear Overhauser effect spectroscopy (NOESY) correlations. Simulated kinetics were used to extrapolate the rate constants of individual diastereomers from the observed rate data, and it correlated well with rate constants measured directly and from isolated diastereomer-enriched samples. The individual diastereomers of a model system fragment at differing rates with half-lives ranging from 5 to 44 min in CDCl3. Density functional theory calculations were performed to investigate the mechanism of fragmentation and support an asynchronous retro-[4 + 2] cycloaddition transition state. The computations generally correlated well with the observed free energies of activation for four diastereomers of the model system as a whole, within 2.6 kcal/mol. However, the observed order of the fragmentation rates across the set of diastereomers deviated from the computational results. YNDs display wide variability in the rate of fragmentation, dependent on the stereoelectronics of the ylidene substituents. A Hammett study showed that the electron-rich aromatic rings attached to the ylidene bridge increase the fragmentation rate, while electron-deficient systems slow fragmentation rates.

Nanoparticle-Decorated Biomimetic Extracellular Matrix for Cell Nanoencapsulation and Regulation.

Cell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell-based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN2 ). This method allows high-capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN2 is a promising platform for advancing cell-based applications.

Venous Anatomy and Collateral Pathways of the Pelvis: An Angiographic Review.

The pelvic venous system is complex, with the potential for numerous pathways of collateralization. Owing to stenosis or occlusion, both thrombotic and nonthrombotic entities in the pelvis may necessitate alternate routes of venous return. Although the pelvic venous anatomy and collateral pathways may demonstrate structural variability, a number of predictable paths often can be demonstrated on the basis of the given disease and the level of obstruction. Several general categories of collateral pathways have been described. These pathway categories include the deep pathway, which is composed of the lumbar and sacral veins and vertebral venous plexuses; the superficial pathway, which is composed of the circumflex and epigastric vessels; various iliofemoral collateral pathways; the intermediate pathway, which is composed of the gonadal veins and the ovarian and uterine plexuses; and portosystemic pathways. The pelvic venous anatomy has been described in detail in cadaveric and anatomic studies, with the aforementioned collateral pathways depicted on CT and MR images in several imaging studies. A comprehensive review of the native pelvic venous anatomy and collateralized pelvic venous anatomy based on angiographic features has yet to be provided. Knowledge of the diseases involving a number of specific pelvic veins is of clinical importance to interventional and diagnostic radiologists and surgeons. The ability to accurately identify common collateral patterns by using multiple imaging modalities, with accurate anatomic descriptions, may assist in delineating underlying obstructive hemodynamics and diagnosing specific occlusive disease entities. ©RSNA, 2022.

A Novel, Open-Source, Low-Cost Bioreactor for Load-Controlled Cyclic Loading of Tendon Explants.

A major risk factor for tendinopathy is tendon overuse (i.e., fatigue loading). Fatigue loading of tendon damages the extracellular matrix and induces tissue degeneration. However, the specific mechanisms linking tendon fatigue damage with tissue degeneration are unclear. While explant models of tendon fatigue loading have been used to address this knowledge gap, they predominantly employ bioreactors that apply cyclic displacements/strains rather than loads/stresses, which are more physiologically relevant. This is because of the technical complexity and cost of building a load-controlled bioreactor, which requires multiple motors, load cells, and computationally intensive feedback loops. Here, we present a novel, low-cost, load-controlled bioreactor that applies cyclic loading to multiple tendon explants by offloading weights from a single motorized stage. Using an optional load cell, we validated that the bioreactor can effectively provide load-controlled fatigue testing of mouse and rat tendon explants while maintaining tissue viability. Furthermore, all the design files, bill of materials, and operating software are available "open source"1 so that anyone can easily manufacture and use the bioreactor for their own research. Therefore, this novel load-controlled bioreactor will enable researchers to study the mechanisms driving fatigue-induced tendon degeneration in a more physiologically relevant and cost-effective manner.

The Elephant in the Cell: Nuclear Mechanics and Mechanobiology.

The 2021 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C) featured a workshop titled "The Elephant in the Room: Nuclear Mechanics and Mechanobiology." The goal of this workshop was to provide a perspective from experts in the field on the current understanding of nuclear mechanics and its role in mechanobiology. This paper reviews the major themes and questions discussed during the workshop, including historical context on the initial methods of measuring the mechanical properties of the nucleus and classifying the primary structures dictating nuclear mechanics, physical plasticity of the nucleus, the emerging role of the linker of nucleoskeleton and cytoskeleton (LINC) complex in coupling the nucleus to the cytoplasm and driving the behavior of individual cells and multicellular assemblies, and the computational models currently in use to investigate the mechanisms of gene expression and cell signaling. Ongoing questions and controversies, along with promising future directions, are also discussed.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates.

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

Analyzing the symmetrical arrangement of structural repeats in proteins with CE-Symm.

Many proteins fold into highly regular and repetitive three dimensional structures. The analysis of structural patterns and repeated elements is fundamental to understand protein function and evolution. We present recent improvements to the CE-Symm tool for systematically detecting and analyzing the internal symmetry and structural repeats in proteins. In addition to the accurate detection of internal symmetry, the tool is now capable of i) reporting the type of symmetry, ii) identifying the smallest repeating unit, iii) describing the arrangement of repeats with transformation operations and symmetry axes, and iv) comparing the similarity of all the internal repeats at the residue level. CE-Symm 2.0 helps the user investigate proteins with a robust and intuitive sequence-to-structure analysis, with many applications in protein classification, functional annotation and evolutionary studies. We describe the algorithmic extensions of the method and demonstrate its applications to the study of interesting cases of protein evolution.

Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Characteristics of studies of pharmacist services registered in ClinicalTrials.gov.

OBJECTIVE: To examine the characteristics of studies of pharmacist services registered in ClinicalTrials.gov. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: ClinicalTrials.gov and MEDLINE databases were searched to identify studies of pharmacist services. Registration information was obtained from the Aggregate Analysis of ClinicalTrials.gov (AACT) database. Studies were excluded if the ClinicalTrials.gov submission date was after December 31, 2018; there were no U.S. study sites; pharmacist services were not mentioned; or pharmacist involvement was limited to medication dispensing, randomization, or measuring study outcomes. OUTCOME MEASURES: Characteristics of registered studies from ClinicalTrials.gov and AACT data, categories of pharmacist services, changes in registration and focus of pharmacist services over time, and relationships between funding source and result availability and between the focus of pharmacist services and types of outcomes and types of pharmacist interventions. RESULTS: Overall 401 studies were identified for initial review, with 151 included for detailed review. Pharmacist services were the only intervention in 68 studies (45.0%), a separate intervention in 14 (9.3%), and part of a combined intervention in 40 (26.5%). In 29 studies (19.2%), pharmacist services were not the focus. Registered studies primarily were interventional, randomized, and open-label; included behavioral or "other" interventions; were conducted in the outpatient setting; and were sponsored by "other" sources. The most common health conditions were hypertension and diabetes. Only 29 of the 104 completed studies (27.9%) posted results. Clinical outcomes were the most common primary (80; 53.0%) and secondary outcomes (66; 58.9%). Medication management (69; 45.7%) and patient education or counseling (88; 58.3%) were the most common types of pharmacist interventions. CONCLUSION: This analysis of ClinicalTrials.gov identified 151 studies of pharmacist services in the United States registered through the end of 2018. Given the breadth of the pharmacy services literature, there is room for improvement in the registration of these types of studies.

Implementing Adapted Individual Placement and Support (IPS) Supported Employment for Transition-Age Youth in Texas.

Transition-age youth (TAY, ages 14-26) diagnosed with serious mental health conditions are at high risk for vocational struggles. This paper examines the implementation and process evaluation of Individual Placement and Support (IPS) and Supported Employment enhanced to better meet developmental needs of TAY. Enhancements include the integration of a TAY development focus, engagement best-practices, Supported Education and Peer Support. Community mental health providers participated in a process evaluation to explore the feasibility of a larger scale implementation. Common organizational barriers were encountered across provider sites including: leadership support, agency structures and funding mechanisms; compounded by the complexity of bridging child and adult systems. Findings have implications for both child and adult community mental health providers as they adapt and integrate programming for TAY.

First report of using low-titer cold-stored type O whole blood in massive postpartum hemorrhage.

BACKGROUND: In cases of massive hemorrhage in the US military, improved outcomes have been reported with the use of warm, fresh whole blood transfusions. Cold-stored low-titer type O whole blood (LTOWB) has become the preferred product for resuscitation of severe bleeding in deployed surgical units. Reports of LTOWB use in civilian trauma are becoming more frequent. CASE REPORT: We report our experience with emergency transfusion of LTOWB for a woman with massive postpartum hemorrhage. The patient had two previous cesarean section deliveries at term without complications. With her third elective cesarean section at term, blood loss during surgery was not excessive, but 3 to 4 hours later she had an estimated blood loss of 3600 mL. Despite measures to control the hemorrhage, she rapidly became hypotensive and tachycardic, and our massive transfusion protocol (MTP) was activated. The transfusion service had very recently incorporated LTOWB into Trauma Pack 1 of the MTP. She received two LTOWB units, after which her hemorrhaging ceased, blood pressure normalized, and she became alert. One hour later she received one unit of fresh frozen plasma and one unit of red blood cells (RBCs). The following morning she received one unit of crossmatched RBCs, for a hematocrit of 20.7%. She was discharged home on Day 4, and she remains healthy. CONCLUSIONS: This is the first report of which we are aware of massive postpartum hemorrhage treated using LTOWB. Our positive experience leads us to speculate that this approach could have a role in massive obstetric hemorrhage.

Pouring of Grains onto Liquid Surfaces: Dispersion or Lump Formation?

This study considers the consequences of adding grains to an air-liquid interface from a funnel. Depending on the grain contact angle and liquid surface tension, the interface is found to support a single or multiple layers of grains, forming a granular stack. By continuing to add grains, the stacks grow until either the lower grains disperse in the liquid, or the complete stack breaks free from the surface and sinks as a dry powder lump. Herein, the effects of grain contact angle, density, and size on these processes are studied experimentally, and a theoretical analysis is given. The maximum number of grains contained in a floating stack and its critical depth are observed to increase as the grain size decreases. The maximum number of grains scales with the bond number (Bo) as Bo-1.82 when stack detachment is observed and with an exponent  -2.0 when grains disperse into the liquid. As a result of these different scaling exponents, a critical bond number above which grains wet and disperse can be identified. Favorable conditions for dispersion are achieved with larger grains and, to a lesser extent, by lower surface tension and contact angle. The critical bond number separating grain dispersion from lump formation increases with an increasing grain contact angle, thus providing a physical justification for increasing grain size with common processes such as granulation or agglomeration. Conversely, a quantitative framework to interpret the limitations in dispersing small grains is proposed, justifying the need for low contact angle or liquids with low surface tensions, both favored by the use of surfactants. The present findings have identified conditions under which lump formation occurs, and hence how these undesired phenomena can be avoided in applications requiring the efficient dispersion of grains across a liquid interface.