FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model.

Fibroblast activation protein (FAP) has attracted considerable attention as a possible target for the radiotherapy of solid tumors. Unfortunately, initial efforts to treat solid tumors with FAP-targeted radionuclides have yielded only modest clinical responses, suggesting that further improvements in the molecular design of FAP-targeted radiopharmaceutical therapies (RPT) are warranted. In this study, we report several advances on the previously described FAP6 radioligand that increase tumor retention and accelerate healthy tissue clearance. Seven FAP6 derivatives with different linkers or albumin binders were synthesized, radiolabeled, and investigated for their effects on binding and cellular uptake. The radioligands were then characterized in 4T1 tumor-bearing Balb/c mice using both single-photon emission computed tomography (SPECT) and ex vivo biodistribution analyses to identify the conjugate with the best tumor retention and tumor-to-healthy organ ratios. The results reveal an optimized FAP6 radioligand that exhibits efficacy and safety properties that potentially justify its translation into the clinic.

Design of a Fibroblast Activation Protein-Targeted Radiopharmaceutical Therapy with High Tumor-to-Healthy-Tissue Ratios.

Because of upregulated expression on cancer-associated fibroblasts, fibroblast activation protein (FAP) has emerged as an attractive biomarker for the imaging and therapy of solid tumors. Although many FAP ligands have already been developed for radiopharmaceutical therapies (RPTs), most suffer from inadequate tumor uptake, insufficient tumor residence times, or off-target accumulation in healthy tissues, suggesting a need for further improvements. Methods: A new FAP-targeted RPT with a novel ligand (FAP8-PEG3-IP-DOTA) was designed by combining the desirable features of several previous ligand-targeted RPTs. Uptake and retention of [111In]In or [177Lu]Lu-FAP8-PEG3-IP-DOTA were assessed in KB, HT29, MDA-MB-231, and 4T1 murine tumor models by radioimaging or ex vivo biodistribution analyses. Radiotherapeutic potencies and gross toxicities were also investigated by monitoring tumor growth, body weight, and tissue damage in tumor-bearing mice. Results: FAP8-PEG3-IP-DOTA exhibited high affinity (half-maximal inhibitory concentration, 1.6 nM) and good selectivity for FAP relative to its closest homologs, prolyl oligopeptidase (half-maximal inhibitory concentration, ∼14.0 nM) and dipeptidyl peptidase-IV (half-maximal inhibitory concentration, ∼860 nM). SPECT/CT scans exhibited high retention in 2 different solid tumor models and minimal uptake in healthy tissues. Quantitative biodistribution analyses revealed tumor-to-healthy-tissue ratios of more than 5 times for all major organs, and live animal studies demonstrated 65%-93% suppression of tumor growth in all 4 models tested, with minimal or no evidence of systemic toxicity. Conclusion: We conclude that [177Lu]Lu-FAP8-PEG3-IP-DOTA constitutes a promising and safe RPT candidate for FAPα-targeted radionuclide therapy of solid tumors.

Monoclonal Antibody chP3R99 Reduces Subendothelial Retention of Atherogenic Lipoproteins in Insulin-Resistant Rats: Acute Treatment Versus Long-Term Protection as an Idiotypic Vaccine for Atherosclerosis.

BACKGROUND: Atherosclerosis is triggered by the retention of apolipoprotein B-containing lipoproteins by proteoglycans. In addition to low-density lipoprotein, remnant lipoproteins have emerged as pivotal contributors to this pathology, particularly in the context of insulin resistance and diabetes. We have previously reported antiatherogenic properties of a monoclonal antibody (chP3R99) that recognizes sulfated glycosaminoglycans on arterial proteoglycans. METHODS AND RESULTS: Solid-phase assays demonstrated that chP3R99 effectively blocked >50% lipoprotein binding to chondroitin sulfate and vascular extracellular matrix in vitro. The preperfusion of chP3R99 (competitive effect) resulted in specific antibody-arterial accumulation and reduced fluorescent lipoprotein retention by ~60% in insulin resistant JCR:LA-cp rats. This competitive reduction was dose dependent (25-250 µg/mL), effectively decreasing deposition of cholesterol associated with lipoproteins. In a 5-week vaccination study in insulin resistant rats with (200 µg subcutaneously, once a week), chP3R99 reduced arterial lipoprotein retention, and was associated with the production of antichondroitin sulfate antibodies (Ab3) able to accumulate in the arteries (dot-blot). Neither the intravenous inoculation of chP3R99 (4.5 mg/kg), nor the immunization with this antibody displayed adverse effects on lipid or glucose metabolism, insulin resistance, liver function, blood cell indices, or inflammation pathways in JCR:LA-cp rats. CONCLUSIONS: Both acute (passive) and long-term administration (idiotypic cascade) of chP3R99 antibody reduced low-density lipoprotein and remnant lipoprotein interaction with proteoglycans in an insulin-resistant setting. These findings support the innovative approach of targeting proatherogenic lipoprotein retention by chP3R99 as a passive therapy or as an idiotypic vaccine for atherosclerosis.

Metabolic inflexibility promotes mitochondrial health during liver regeneration.

Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial ß-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.

Critical analysis of polycyclic tetramate macrolactam biosynthetic gene cluster phylogeny and functional diversity.

Polycyclic tetramate macrolactams (PTMs) are bioactive natural products commonly associated with certain actinobacterial and proteobacterial lineages. These molecules have been the subject of numerous structure-activity investigations since the 1970s. New members continue to be pursued in wild and engineered bacterial strains, and advances in PTM biosynthesis suggest their outwardly simplistic biosynthetic gene clusters (BGCs) belie unexpected product complexity. To address the origins of this complexity and understand its influence on PTM discovery, we engaged in a combination of bioinformatics to systematically classify PTM BGCs and PTM-targeted metabolomics to compare the products of select BGC types. By comparing groups of producers and BGC mutants, we exposed knowledge gaps that complicate bioinformatics-driven product predictions. In sum, we provide new insights into the evolution of PTM BGCs while systematically accounting for the PTMs discovered thus far. The combined computational and metabologenomic findings presented here should prove useful for guiding future discovery.IMPORTANCEPolycyclic tetramate macrolactam (PTM) pathways are frequently found within the genomes of biotechnologically important bacteria, including Streptomyces and Lysobacter spp. Their molecular products are typically bioactive, having substantial agricultural and therapeutic interest. Leveraging bacterial genomics for the discovery of new related molecules is thus desirable, but drawing accurate structural predictions from bioinformatics alone remains challenging. This difficulty stems from a combination of previously underappreciated biosynthetic complexity and remaining knowledge gaps, compounded by a stream of yet-uncharacterized PTM biosynthetic loci gleaned from recently sequenced bacterial genomes. We engaged in the following study to create a useful framework for cataloging historic PTM clusters, identifying new cluster variations, and tracing evolutionary paths for these molecules. Our data suggest new PTM chemistry remains discoverable in nature. However, our metabolomic and mutational analyses emphasize the practical limitations of genomics-based discovery by exposing hidden complexity.

Piperazate-Guided Isolation of Caveamides A and B, Cyclohexenylalanine-Containing Nonribosomal Peptides from a Cave Actinomycete.

Hybrid genome-mining/15N-NMR was used to target compounds containing piperazate (Piz) residues, leading to the discovery of caveamides A (1) and B (2) from Streptomyces sp. strain BE230, isolated from New Rankin Cave (Missouri). Caveamides are highly dynamic molecules containing an unprecedented ß-ketoamide polyketide fragment, two Piz residues, and a new N-methyl-cyclohexenylalanine residue. Caveamide B (2) exhibited nanomolar cytotoxicity against several cancer cell lines and nanomolar antimicrobial activity against MRSA and E. coli.

Hypotensive drugs mitigate the high-sodium diet-induced pro-inflammatory activation of mouse macrophages in vivo.

Nowadays, there is an increasing emphasis on the need to alleviate the chronic inflammatory response to effectively treat hypertension. However, there are still gaps in our understanding on how to achieve this. Therefore, research on interaction of antihypertensive drugs with the immune system is extremely interesting, since their therapeutic effect could partly result from amelioration of hypertension-related inflammation, in which macrophages seem to play a pivotal role. Thus, current comprehensive studies have investigated the impact of repeatedly administered hypotensive drugs (captopril, olmesartan, propranolol, carvedilol, amlodipine, verapamil) on macrophage functions in the innate and adaptive immunity, as well as if drug-induced effects are affected by a high-sodium diet (HSD), one of the key environmental risk factors of hypertension. Although the assayed medications increased the generation of reactive oxygen and nitrogen intermediates by macrophages from standard fed donors, they reversed HSD-induced enhancing effects on macrophage oxidative burst and secretion of pro-inflammatory cytokines. On the other hand, some drugs increased macrophage phagocytic activity and the expression of surface markers involved in antigen presentation, which translated into enhanced macrophage ability to activate B cells for antibody production. Moreover, the assayed medications augmented macrophage function and the effector phase of contact hypersensitivity reaction, but suppressed the sensitization phase of cell-mediated hypersensitivity under HSD conditions. Our current findings contribute to the recognition of mechanisms, by which excessive sodium intake affects macrophage immune activity in hypertensive individuals, and provide evidence that the assayed medications mitigate most of the HSD-induced adverse effects, suggesting their additional protective therapeutic activity.

Preoperative Multisensory Room Use in Pediatric Patients With Autism: A Randomized Clinical Trial.

Purpose: To evaluate the impact of multisensory room (MSR) use on preoperative anxiety and postoperative outcomes in children with autism spectrum disorder (ASD) undergoing dental treatment with general anesthesia. Methods: Forty children, ages six to 17 years, with ASD re- quiring general anesthesia for dental treatment, participated in this study. Participants were randomized to either the control group (standard pre- operative waiting room) or intervention group (MSR) for 20 minutes prior to general anesthesia induction. Pre- and post-intervention preoperative anxiety were measured. Following surgery, postoperative emergence delirium was assessed. Short- and long-term postoperative pain and adverse behavioral effects were evaluated six hours, 24 hours, one week, and one month post-surgery. Data analysis employed repeated measures analysis of variance with two groups and either two or four time periods. Results: The sample was predominantly male (62.5 percent) and identified as either White or Black (53 percent) and non-Hispanic (60 percent). Preoperative behavioral anxiety levels increased post-intervention in the control group (P<0.05) and decreased in the MSR group (P<0.001). Following surgery, pain intensity was greater in the control group compared to the MSR group at six hours (P<0.05) and 24 hours (P<0.01), and similar at one and four weeks. Pre- and post-intervention measures of preoperative heart rate, postoperative emergence delirium, and behavioral effects were similar between groups and over time. Conclusion: These findings suggest a novel, nonpharmacologic technique that can be utilized by various health care specialties to reduce preoperative anxiety and improve post- operative outcomes in children with autism spectrum disorder.

Poliovirus-Neutralizing Antibody Seroprevalence and Vaccine Habits in a Vaccine-Derived Poliovirus Outbreak Region in the Democratic Republic of Congo in 2018: The Impact on the Global Eradication Initiative.

Despite the successes in wild-type polio eradication, poor vaccine coverage in the DRC has led to the occurrence of circulating vaccine-derived poliovirus outbreaks. This cross-sectional population-based survey provides an update to previous poliovirus-neutralizing antibody seroprevalence studies in the DRC and quantifies risk factors for under-immunization and parental knowledge that guide vaccine decision making. Among the 964 children between 6 and 35 months in our survey, 43.8% (95% CI: 40.6-47.0%), 41.1% (38.0-44.2%), and 38.0% (34.9-41.0%) had protective neutralizing titers to polio types 1, 2, and 3, respectively. We found that 60.7% of parents reported knowing about polio, yet 25.6% reported knowing how it spreads. Our data supported the conclusion that polio outreach efforts were successfully connecting with communities-79.4% of participants had someone come to their home with information about polio, and 88.5% had heard of a polio vaccination campaign. Additionally, the odds of seroreactivity to only serotype 2 were far greater in health zones that had a history of supplementary immunization activities (SIAs) compared to health zones that did not. While SIAs may be reaching under-vaccinated communities as a whole, these results are a continuation of the downward trend of seroprevalence rates in this region.

Heterogeneity of Size and Toxin Distribution in Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium associated with localized aggressive periodontitis as well as some systemic diseases. The strains of A. actinomycetemcomitans most closely associated with disease produce more of a secreted leukotoxin (LtxA) than isolates from healthy carriers, suggesting a key role for this toxin in disease progression. LtxA is released into the bacterial cytosol in a free form as well as in association with the surface of outer membrane vesicles (OMVs). We previously observed that the highly leukotoxic A. actinomycetemcomitans strain JP2 produces two populations of OMVs: a highly abundant population of small (<100 nm) OMVs and a less abundant population of large (>300 nm) OMVs. Here, we have developed a protocol to isolate the OMVs produced during each specific phase of growth and used this to demonstrate that small OMVs are produced throughout growth and lack LtxA, while large OMVs are produced only during the exponential phase and are enriched with LtxA. Our results indicate that surface-associated DNA drives the selective sorting of LtxA into large OMVs. This study provides valuable insights into the observed heterogeneity of A. actinomycetemcomitans vesicles and emphasizes the importance of understanding these variations in the context of bacterial pathogenesis.

Predictive utility of remnant cholesterol in atherosclerotic cardiovascular disease.

PURPOSE OF REVIEW: Remnant cholesterol (RC) is the cholesterol carried in lipoproteins derived from the catabolism of chylomicrons and very low-density lipoproteins. Evidence supporting the causal relationship of RC with atherosclerotic cardiovascular disease (ASVD) is accumulating rapidly. The number of impactful contributions to this field are increasing and provide a pathophysiological insight into the current residual cardiovascular risk beyond low-density cholesterol (LDL)-cholesterol (LDL-C). They also raise the question of whether RC should be used in prediction models and become the target of new therapeutic interventions. The intent of this review is to highlight the recent advances on the role of RC in atherogenesis and the validation of RC as a predictor of ASVD. RECENT FINDINGS: Numerous prospective and retrospective cohorts helped validate a significant causal relationship of RC with various forms of ASVD, independent of LDL-C. A recent large Mendelian randomization study reinforced the existence of this relationship and showed that the risk of atherosclerotic events was driven nearly entirely by a direct effect of RC. SUMMARY: Both available and accumulating evidence suggest that a lifelong reduction in RC could translate into a substantial reduction in ASVD risk. The data support a revision of current guidelines to incorporate RC as an independent risk factor for ASVD. We propose that early screening of RC should be implemented and that RC lowering should become the target of future drug developments.

Beneficial Effects of Dietary Flaxseed on Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), a significant cause of chronic liver disease, presents a considerable public health concern. Despite this, there is currently no treatment available. This study aimed to investigate dietary flaxseed in the JCR:LA-corpulent rat strain model of NAFLD. Both obese male and female rats were studied along with their lean counterparts after 12 weeks of ingestion of a control diet, or control diet with flaxseed, or high fat, high sucrose (HFHS), or HFHS plus flaxseed. Obese rats showed higher liver weight and increased levels of cholesterol, triglyceride, and saturated fatty acid, which were further elevated in rats on the HFHS diet. The HFHS diet induced a significant two-fold elevation in the plasma levels of both aspartate aminotransferase and alanine aminotransferase in the obese male and female rats. Including flaxseed in the HFHS diet significantly lowered liver weight, depressed the plasma levels of both enzymes in the obese male rats, and reduced hepatic cholesterol and triglyceride content as well as improving the fatty acid profile. In summary, including flaxseed in the diet of male and female obese rats led to an improved lipid composition in the liver and significantly reduced biomarkers of tissue injury despite consuming a HFHS chow.

Rapid exchange cooling with trapped ions.

The trapped-ion quantum charge-coupled device (QCCD) architecture is a leading candidate for advanced quantum information processing. In current QCCD implementations, imperfect ion transport and anomalous heating can excite ion motion during a calculation. To counteract this, intermediate cooling is necessary to maintain high-fidelity gate performance. Cooling the computational ions sympathetically with ions of another species, a commonly employed strategy, creates a significant runtime bottleneck. Here, we demonstrate a different approach we call exchange cooling. Unlike sympathetic cooling, exchange cooling does not require trapping two different atomic species. The protocol introduces a bank of "coolant" ions which are repeatedly laser cooled. A computational ion can then be cooled by transporting a coolant ion into its proximity. We test this concept experimentally with two 40Ca+ ions, executing the necessary transport in 107 µs, an order of magnitude faster than typical sympathetic cooling durations. We remove over 96%, and as many as 102(5) quanta, of axial motional energy from the computational ion. We verify that re-cooling the coolant ion does not decohere the computational ion. This approach validates the feasibility of a single-species QCCD processor, capable of fast quantum simulation and computation.

Natural history of adenoviral conjunctivitis in a US-based population: Viral load, signs, and symptoms.

PURPOSE: To report the clinical signs, symptoms, and viral clearance in individuals in the United States with adenoviral conjunctivitis (Ad-Cs). METHODS: Individuals ≥ 18 years presenting within 4 days of symptoms of Ad-Cs who met eligibility criteria and tested positive with both point-of-care immunoassay antigen and quantitative polymerase chain reaction (qPCR) testing were enrolled. Patient-reported symptoms, clinician-graded signs, and qPCR viral titers were collected at baseline, days 1-2, 4 (days 3-5), 7 (days 6-10), 14 (days 11-17) and 21 (days 18-21). RESULTS: There was no detectable viral titers by the day 14 visit in 6/8 patients. By day 21, there was no detectable viral titers in the 7 participants who completed the visit; however, signs and symptoms persisted including: blurry vision (5/7), discomfort (2/7) or redness (1/7). Masked clinicians also noted conjunctival redness (4/7), follicular conjunctivitis (4/7) and bulbar edema (3/7). CONCLUSION: Many patient-reported symptoms and clinical signs persist after viral titers are no longer detectable by qPCR. Using clinical signs and symptoms to determine quarantine duration may result in patients being furloughed longer than the time that the patient is infectious.

Muscle Fiber, Connective Tissue and Meat Quality Characteristics of Pork from Low Birth Weight Pigs as Affected by Diet-Induced Increased Fat Absorption and Preferential Muscle Marbling.

This study investigated how birth weight differences in piglets affected carcass and muscle fiber properties as well as meat quality at slaughter. Within litters, piglets were grouped according to their birth weight as either normal (NBW; 1.62-1.73 kg) or low (LBW; 1.18-1.29 kg). At 5 weeks of age, NBW piglets were randomly transitioned to control (C) or isocaloric high fat diets derived from non-dairy (HF), while LBW piglets were randomly transitioned to high fat diets derived from non-dairy (HF) or dairy sources (HFHD). Piglets were reared in individual pens under standardized housing and feeding conditions. Live weight was recorded weekly, and pigs were slaughtered at 12 weeks of age. Hot carcass weights, dressing percentages, lean meat yield, and primal cut proportions were determined. The m. longissimus thoracis was collected from the right side of the carcass for measurement of physical and chemical properties of meat and muscle fiber characteristics. Results indicated that LBW pigs compensated for their live weight compared to NBW pigs at 6 weeks of age. The mean muscle fiber diameter of LBW-HFHD group is significantly higher than NBW-C and NBW-HF group, and the type I muscle fiber diameter is significantly higher than NBW-C group. Dairy fat inclusion in LBW pig diet reduced carcass back fat thickness. This increased the calculated lean meat yield to be comparable to that of NBW pigs fed a commercial diet. Incorporating dairy-sourced high-fat into LBW pigs' diets appears to be an effective strategy for producing carcasses equivalent to NBW pigs.

PRAME Immunohistochemistry for Distinguishing Vulvar and Vaginal Melanoma From Benign Melanocytic Nevi.

Vulvovaginal melanoma (VVM) is a rare but deadly disease, accounting for 5% of all vulvar malignancies, with a 5-yr survival rate of only 47% for all stages of the disease. VVM is a distinct subset of melanoma, with a unique genomic profile and underlying pathogenesis unassociated with sun exposure. Distinguishing these rare malignancies from very common pigmented lesions of the vulva and vagina is challenging as histologic features often overlap between entities. PReferentially expressed Antigen in MElanoma (PRAME) is a melanoma-associated protein, and immunohistochemistry (IHC) for PRAME distinguishes cutaneous, oral mucosal, and retinal melanoma from atypical nevi. Given the biological differences between VVM and cutaneous melanoma, the utility of PRAME IHC for the diagnosis of VVM is unknown. We accrued a cohort of 20 VVM and 21 benign vulvar melanocytic nevi. We found that nuclear PRAME IHC staining with 4+ intensity was present in 85% of the VVM and 0% of the nevi. With the assistance of PRAME IHC, we found evidence of close or positive margin involvement in 3 of 10 cases where margins were originally diagnosed as negative for melanoma in situ. Our study is the first to assess PRAME IHC in a cohort of VVM cases and provides confidence for using PRAME IHC to assist with diagnosis and margin assessment in this rare disease.

Evaluating the accuracy of a cataract surgery simulation video in depicting patient experiences under conscious anesthesia.

PURPOSE: To evaluate the accuracy of a point-of-view cataract surgery simulation video in representing different subjective experiences of patients undergoing the procedure. METHODS: One hundred consecutive post-cataract-surgery patients were shown a short simulation video of the surgery obtained through a porcine eye model during the first postoperative week. Patients then answered a multiple-choice questionnaire regarding their visual and tactile intraoperative experiences and how those experiences matched the simulation. RESULTS: Of the patients surveyed (n = 100), 78% (n = 78) recalled visual experiences during surgery, 11% recalled pain (n = 11), and 6.4% (n = 5) recalled frightening experiences. Thirty-six percent of patients (n = 36) were interviewed after their second cataract surgery; there was no statistically significant difference between anxiety scores reported before the first eye surgery and second eye surgery (p = 0.147). Among all patients who recalled visual experiences (n = 78), nearly half (47.4%) reported that the video was the same/similar to their experience. Forty-eight percent of the patients recommended future patients to watch the video before their procedures, and more than a third (36%) agreed that watching the video before surgery would have helped them to relax. CONCLUSIONS: Our model reflects the wide range of subjective patient experiences during and after surgery. The high percentage of patients who found the video accurate in different ways suggests that, with more development, point-of-view cataract simulation videos could prove useful for educational or clinical use. Further research may be done to confirm the simulation's utility, by screening the video for subjects before operations.