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Food allergy, particularly peanut allergy (PA), is a growing health concern affecting millions globally. PA can lead to severe reactions, including fatal anaphylaxis. Despite the availability of FDA-approved therapies like Palforzia, a cure remains elusive. Current immunotherapies show promise but lack a definitive cure. This study applies an established computational biology tool to design aptamers targeting Ara h1 and Ara h2. The in silico design aims to streamline the selection process, enabling cost-effective and rapid identification of aptamer candidates. The developed aptamers (AYA22A, including AYA22AR321, AYA22AR211, and AYA22AR524), demonstrated efficacy in inhibiting degranulation of RBL-2H3 cells (rat basophilic leukemia cell line) in vitro. They showed promise in neutralizing peanut allergen-induced immune responses. The selected aptamers inhibited degranulation in RBL-2H3 cells, addressing concerns in raw peanuts. Moreover, these aptamers demonstrated stability and effectiveness in peanut plant seeds and commercial products. Our aptamers exhibited potential in modulating immune responses associated with peanut allergy. They influenced Th1/Th2 balance, indicating a role in cytokine regulation. In vitro studies also showed the aptamers’ impact on immune cell expression and cytokine production, resembling responses observed with established immunotherapies. The findings suggest AYA22A aptamers as a potential therapeutic option for peanut allergy, providing a basis for further in vivo investigations.

The nasal microenvironment plays a crucial role in the transmission, modulation, and clinical progression of COVID-19; however, the immune responses at the site of viral entry remain poorly understood. We deciphered the link between nasopharyngeal (NP) immune and inflammatory response that triggers cytokine/chemokine storms in the nasal route of COVID-19-positive patients. We used RT-PCR, multiplex ELISA, flow cytometry, and LC-MS/MS to decipher nasopharyngeal immune perturbations associated with severe COVID-19. In addition, we performed in vitro assays using cultured human monocytes-derived macrophages trained both in the presence and absence of SARS-CoV-2 trimeric spike protein(s) and co-cultured with and without autologous human peripheral blood mononuclear cells (hPBMCs)/total T-cells/CD8 T-cells. In vitro immune perturbations were examined by flow cytometry and LC-MS/MS assays. Our findings confirm that macrophages orchestrate NP immune inflammatory responses and highlight the cytokine/chemokine storms associated with the increased CD8+T-cells along with Tregs, Th1, and Th17.1 T-helper cells. We observed a correlation between in vitro and nasal findings that trained macrophages, profoundly M2c, differentially promote the inflammatory surfactome on CD8 T-cells, including ITGAM, LGALS3, CD38, TKT, LRPAP1, and SSBP1. The findings of this study conclude that inflammatory lymphocyte perturbations within the nasopharynx of COVID-19 patients may enforce immune homeostasis during SARS-CoV-2-infection and contribute to COVID-19 pathology. This study explored the therapeutic target proteins that could facilitate the development of new medications, which could allow for immediate treatment of possible emerging viral infections.

The continuous emergence of new variants of concern for SARS-CoV-2 has created a challenge for existing therapies. To address this, we developed a series of single-stranded DNA aptamers that not only bind specifically to the trimer S protein of SARS-CoV-2 but also block the interaction between the trimer S protein and ACE2 receptors. The systematic evolution of ligands by exponential enrichment (SELEX) was performed to select the aptamers for SARS-CoV-2 trimer S protein. ELISA-based assay and flow cytometry were performed to test the apatmers’ binding and inhibition of trimer S protein in vitro. Binding affinity was measured using surface plasmon resonance. Significance was determined in Prism 9.0 using the one-way ANOVA test (Dunnett’s multiple comparisons test) or two-way ANOVA test (Tukey’s multiple comparisons test) for comparisons. The p values < 0.05 were considered statistically significant. After 12 rounds of SELEX, eight highly enriched aptamers were able to bind to the trimer S protein of the SARS-CoV-2 Wuhan original strain as well as the trimer S proteins of the Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants, with affinities in the nM range, while also inhibiting their interaction with ACE2 receptors in Vero E6 cells. Modifications to our best aptamer were made by adding forward and reverse primer sequences and truncation. The modified aptamers AYA2012004_L and AYA2012004_L-M1 showed up to 70% inhibition of the binding of virus-like particles (VLPs) expressing S protein to the ACE2 receptor expressed in HEK293T cells. Our findings imply that the selected aptamers can prevent SARS-CoV-2 from entering host cells and hence suppress the viral infection. In addition, the findings suggest that the selected aptamers might be an innovative therapy for the treatment of COVID-19.

Abstract Background The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. Methods In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. Results We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. Conclusions Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Generative models for the novo molecular design attract enormous interest for exploring the chemical space. Here the authors investigate the application of chemical language models to challenging modeling tasks demonstrating their capability of learning complex molecular distributions.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Background Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR T) therapy showed remarkable efficacy in patients with relapsed or refractory multiple myeloma (RRMM). This phase 1 dose-escalation and expansion study developed C-CAR088, a novel second-generation humanized anti-BCMA CAR T-cell therapy, and assessed the safety and efficacy of three dosages of C-CAR088 in patients with RRMM.Methods Patients received lymphodepletion with three doses of cyclophosphamide (300 mg/m2) and three doses of fludarabine (30 mg/m2) on days –5, –4, and –3, followed by an infusion of C-CAR088 on day 0. Doses of 1.0×106, 3.0×106, and 6.0×106 CAR T cells/kg (±20%) were tested in the dose-escalation cohorts and expansion cohorts. The primary endpoint was treatment safety, including the rate of treatment-emergent adverse events after cell infusion. Secondary endpoints were the overall response rate and progression-free survival. The exploratory endpoints were the quantification of C-CAR088 CAR T cells, selection of cytokines and chemokines in blood, and measurement of tumor BCMA expression.Results As of July 2, 2021, 31 patients had been infused with C-CAR088. Any grade cytokine release syndrome (CRS) occurred in 29 patients (93.5%), and grade 3 CRS occurred in 3 patients (9.7%). One patient from the high-dose group (4.5–6.0×106 CAR T cells/kg) developed grade 1 neurotoxicity. No dose-limiting toxicities were observed in any dose group, and all adverse events were reversible after proper management. The overall response, stringent complete response, complete response (CR), and very good partial response rates were 96.4%, 46.4%, 10.7%, and 32.1%, respectively. The CR rate in the medium-dose (3.0×106 CAR T cells/kg) and high-dose (4.5–6.0×106 CAR T cells/kg) groups was 54.5% and 71.4%, respectively. In the CR group, 15 (93.7%) patients achieved minimal residual disease (MRD) negativity (test sensitivity >1/10−5). All seven patients with double-hit or triple-hit multiple myeloma achieved MRD-negative CR.Conclusions The present study demonstrated that C-CAR088 had a good safety profile and high antitumor activity in patients with RRMM, constituting a promising treatment option for RRMM.Trial registration number NCT03815383, NCT03751293, NCT04295018, and NCT04322292.

Serum or plasma have been the primary focus of proteomics studies for COVID-19 to identity biomarkers and potential drug targets. The nasal mucosal environment which consists of lipids, mucosal immune cells, and nasal proteome, has been largely neglected but later revealed to have critical role combating SARS-CoV-2 infection. We present a bottom-up proteomics investigation of the host response to SARS-CoV-2 infection in the nasopharyngeal environment, featuring a noninvasive approach using proteins in nasopharyngeal swabs collected from groups of 76 SARS-CoV-2 positive and 76 negative patients. Results showed that 31 significantly down-regulated and 6 up-regulated proteins were identified (p < 0.05, log2 FC > 1.3) in SARS-CoV-2 positive patient samples as compared to the negatives; these proteins carry potential value as markers for the early detection of COVID-19, disease monitoring, as well as be drug targets. The down-regulation of coagulation factor 5 indicates a thrombotic abnormality in COVID-19 patients and the decreased IgG4 suggests an abnormal immune response at the point of entry in human nasopharyngeal environment, which is in consistent with KEGG and GO pathway analysis. Our study also demonstrated that mass spectrometry proteomics analysis of nasopharyngeal swabs can be used as a powerful early approach to evaluate host response to SARS-CoV-2 viral infection.

Background. Radiation after resection of an atypical lipomatous tumor (ALT) is controversial. This study evaluates local control and complications after the first resection of ALTs of the extremity with or without adjuvant radiation. Methods. A dual institution, retrospective review of patients treated from 1995 to 2020 with first-time resection of an ALT in the extremity was performed. In total, 102 patients underwent adjuvant radiation (XRT group) and 68 patients were treated with surgery alone (no-XRT group). The median follow-up time was 4.6 years (interquartile range (IQR) 2.0–7.3 years). The median radiation dose was 60 Gy (IQR 55–66 Gy). Univariable and multivariable analyses evaluated the association of patient, tumor, and treatment variables with recurrence and complications. Kaplan–Meier analysis evaluated local recurrence-free survival (LRFS) and time to complication. Results. The overall incidence of local recurrence was 1% (1/102) in the XRT group and 24% (16/68) in the no-XRT group p

Abstract Directed differentiation of human pluripotent stem cells (hPSCs) has enabled the generation of specific neuronal subtypes that approximate the intended primary mammalian cells on both the RNA and protein levels. These cells offer unique opportunities, including insights into mechanistic understanding of the early driving events in neurodegenerative disease, replacement of degenerating cell populations, and compound identification and evaluation in the context of precision medicine. However, whether the derived neurons indeed recapitulate the physiological features of the desired bona fide neuronal subgroups remains an unanswered question and one important for validating stem cell models as accurate functional representations of the primary cell types. Here, we purified both hPSC-derived and primary mouse spinal motor neurons in parallel and used extracellular multi-electrode array (MEA) recording to compare the pharmacological sensitivity of neuronal excitability and network function. We observed similar effects for most receptor and channel agonists and antagonists, supporting the consistency between human PSC-derived and mouse primary spinal motor neuron models from a physiological perspective.

Abstract Background With over 3 million US prostate cancer survivors, ensuring high‐quality, coordinated cancer survivorship care is important. However, implementation of recommended team‐based cancer care has lagged, and determinants of quality care across primary and specialty care remain unclear. Guided by the theoretical domains framework (TDF), we explored multidisciplinary determinants of quality survivorship care in an integrated delivery system. Methods We conducted semistructured interviews with primary (4) and specialty (7) care providers across 6 Veterans Health Administration clinic sites. Using template analysis, we coded interview transcripts into the TDF, mapping statements to specific constructs within each domain. We assessed whether each construct was perceived a barrier or facilitator, examining results for both primary care providers (PCPs) and prostate cancer specialists. Results Cancer specialists and PCPs identified 2 primary TDF domains impacting their prostate cancer survivorship care: Knowledge and Environmental context and resources. Both groups noted knowledge (about survivorship care) and procedural knowledge (about how to deliver survivorship care) as positive determinants or facilitators, whereas resources/material resources (to deliver survivorship care) was noted as a negative determinant or barrier to care. Additional domains more commonly referenced by cancer specialists included Social/professional role and identity and Goals, while PCPs reported the domain Beliefs about capabilities as relevant. Conclusions We used the TDF to identify several behavioral domains acting as determinants of high‐quality, team‐based prostate cancer survivorship care. These results can inform prostate cancer survivorship care plan content, and may guide tailored, multidisciplinary implementation strategies to improve survivorship care across the primary and specialty care interface.

Summary: High-throughput physiological assays lose single-cell resolution, precluding subtype-specific analyses of activation mechanism and drug effects. We demonstrate APPOINT (automated physiological phenotyping of individual neuronal types), a physiological assay platform combining calcium imaging, robotic liquid handling, and automated analysis to generate physiological activation profiles of single neurons at large scale. Using unbiased techniques, we quantify responses to sequential stimuli, enabling subgroup identification by physiology and probing of distinct mechanisms of neuronal activation within subgroups. Using APPOINT, we quantify primary sensory neuron activation by metabotropic receptor agonists and identify potential contributors to pain signaling. We expand the role of neuroimmune interactions by showing that human serum directly activates sensory neurons, elucidating a new potential pain mechanism. Finally, we apply APPOINT to develop a high-throughput, all-optical approach for quantification of activation threshold and pharmacologically validate contributions of ion channel families to optical activation. Motivation: Physiological assays are typically small scale, such as patch clamp and traditional calcium imaging, whereas larger-scale techniques lose cellular resolution and thus have limited value in heterogeneous neuronal populations. Applications that require analysis of large cell numbers, including screens or experiments that address neuronal diversity, require larger-scale physiological characterization at single-cell resolution. In addition, small-scale tools are limited by experimental and analytical biases, and the desired platform would reduce these biases. We combine high-content longitudinal calcium imaging with liquid handling and an unbiased machine-learning-based analysis pipeline to generate a tool for larger-scale granular physiological investigation.

Abstract Background Men with prostate cancer are often castrated with long-acting injectable drugs termed androgen deprivation therapy (ADT). Although many benefit, ADT is also used in patients with little or nothing to gain. The best ways to stop this practice are unknown, and range from blunt pharmacy restrictions to informed decision-making. This study will refine and pilot two different de-implementation strategies for reducing ADT use among those unlikely to benefit in preparation for a comparative effectiveness trial. Methods/design This innovative mixed methods research program has three aims. Aim 1: To assess preferences and barriers for de-implementation of chemical castration in prostate cancer. Guided by the theoretical domains framework (TDF), urologists and patients from facilities with the highest and lowest castration rates across the VA will be interviewed to identify key preferences and de-implementation barriers for reducing castration as prostate cancer treatment. This qualitative work will inform Aim 2 while gathering rich information for two proposed pilot intervention strategies. Aim 2: To use a discrete choice experiment (DCE), a novel barrier prioritization approach, for de-implementation strategy tailoring. The investigators will conduct national surveys of urologists to prioritize key barriers identified in Aim 1 for stopping incident castration as localized prostate cancer treatment using a DCE experiment design. These quantitative results will identify the most important barriers to be addressed through tailoring of two pilot de-implementation strategies in preparation for Aim 3 piloting. Aim 3: To pilot two tailored de-implementation strategies to reduce castration as localized prostate cancer treatment. Building on findings from Aims 1 and 2, two de-implementation strategies will be piloted. One strategy will focus on formulary restriction at the organizational level and the other on physician/patient informed decision-making at different facilities. Outcomes will include acceptability, feasibility, and scalability in preparation for an effectiveness trial comparing these two widely varying de-implementation strategies. Discussion Our innovative approach to de-implementation strategy development is directly aligned with state-of-the-art complex implementation intervention development and implementation science. This work will broadly advance de-implementation science for low value cancer care, and foster participation in our de-implementation evaluation trial by addressing barriers, facilitators, and concerns through pilot tailoring. Trial registration ClinicalTrials.gov Identifier: NCT03579680, First Posted July 6, 2018.

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Sensory neuron hyperexcitability is a critical driver of pathological pain and can result from axon damage, inflammation, or neuronal stress. G-protein coupled receptor signaling can induce pain amplification by modulating the activation of Trp-family ionotropic receptors and voltage-gated ion channels. Here, we sought to use calcium imaging to identify novel inhibitors of the intracellular pathways that mediate sensory neuron sensitization and lead to hyperexcitability. We identified a novel stimulus cocktail, consisting of the SSTR2 agonist L-054,264 and the S1PR3 agonist CYM5541, that elicits calcium responses in mouse primary sensory neurons in vitro as well as pain and thermal hypersensitivity in mice in vivo. We screened a library of 906 bioactive compounds and identified 24 hits that reduced calcium flux elicited by L-054,264/CYM5541. Among these hits, silymarin, a natural product derived from milk thistle, strongly reduced activation by the stimulation cocktail, as well as by a distinct inflammatory cocktail containing bradykinin and prostaglandin E2. Silymarin had no effect on sensory neuron excitability at baseline, but reduced calcium flux via Orai channels and downstream mediators of phospholipase C signaling. In vivo, silymarin pretreatment blocked development of adjuvant-mediated thermal hypersensitivity, indicating potential use as an antiinflammatory analgesic. [ABSTRACT FROM AUTHOR]