Free Release Research Articles

Evaluation of the cell death markers for aberrated cell free DNA release in high altitude pulmonary edema.

The effect of high altitude (HA, altitude >2500 m) can trigger a maladaptive response in unacclimatized individuals, leading to various HA illnesses such as high altitude pulmonary edema (HAPE). The present study investigates circulating cell free (cf) DNA, a minimally invasive biomarker that can elicit a pro-inflammatory response. Our earlier study observed altered cfDNA fragment patterns in HAPE patients and the significant correlation of these patterns with peripheral oxygen saturation levels. However, the unclear release mechanisms of cfDNA in circulation limit its characterization and clinical utility. The present study not only observed a significant increase in cfDNA levels in HAPE patients (27.03 ± 1.37 ng/ml; n = 145) compared to healthy HA sojourners (controls, 14.57 ± 0.74 ng/ml; n = 65) and highlanders (HLs, 15.50 ± 0.8 ng/ml; n = 34) but also assayed the known cell death markers involved in cfDNA release at HA. The study found significantly elevated levels of the apoptotic marker, annexin A5, and secondary necrosis or late apoptotic marker, High mobility group box 1, in HAPE patients. In addition, we observed ahigher oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine, in HAPE compared to controls, suggestive of the role of oxidative DNA status in promoting the inflammatory potential of cfDNA fragments and their plausible role in manifesting HAPE pathophysiology. Extensive in vitro future assays can confirm theimmunogenic role of cfDNA fragments that may act as a danger-associated molecular pattern and associate with markers of cellular stresses in HAPE.

Enhanced Oligopeptide and Free Tryptophan Release from Chickpea and Lentil Proteins: A Comparative Study of Enzymatic Modification with Bromelain, Ficin, and Papain.

Plant-based foods offer a sustainable alternative to meet the growing protein demand. Legumes are the most promising of these, as they contain relatively high concentrations of protein, low digestible starch, and dietary fiber, as well as them possibly featuring low levels of fat. Enzymatically modified legume proteins provide us with tempting perspectives in terms of enhancing foods' biological values. However, their bioavailability and digestibility are generally less sufficient than that of proteins of animal origin, which may be improved by well-tailored enzyme modification. In this study, the efficacy of three plant-based proteases (bromelain, ficin, and papain) were evaluated at two distinct concentrations (2.5% and 10%) and three hydrolysis durations (1, 2, and 12 h) when transforming chickpea and lentil proteins. The degree of hydrolysis (DH), peptide profiles, and free amino acid content were analyzed to determine the efficiency of each enzyme. Results showed significant variations in DH, which was influenced by enzyme type, concentration, and hydrolysis duration. Papain exhibited the highest DH, particularly at a 10% concentration, reaching 27.8% efficiency in chickpea and 34.8% in lentils after 12 h. Bromelain and ficin were proven to be less effective, with ficin showing the least hydrolytic activity. SDS-PAGE analysis revealed substantial protein degradation, especially subsequent to papain treatment, pointing out that most proteins were cleaved into smaller peptides. SEC-HPLC indicated a predominant release of peptides within the 200-1000 Da range, suggesting enhanced bioavailability. Papain and bromelain treatments resulted in a significant release of oligopeptides and dipeptides. UHPLC analysis highlighted a marked post-hydrolysis increase in total free amino acids, with arginine, leucine, and lysine being the most abundant ones. Notably, tryptophan, being undetectable in untreated samples, was released in measurable amounts post-hydrolysis. These findings demonstrate papain's superior performance in protein hydrolysis and its potential in producing bioactive peptides, highlighting its applicability in food processing and the development of both nutraceuticals and functional foods.

The impact of heating-induced lactosylation on the digestibility of lactotransferrin

Lactosylation of lysines occurs during the heating of dairy products, yet how lactosylation impact the lysine release during digestion remains largely unknown. This study examines the effect of lactosylation on the digestibility of lactotransferrin using chemical analysis, proteomics, and peptidomics. Under the applied heating conditions, lactotransferrin primarily undergoes early-stage Maillard reactions, producing lactulose-lysine. Furosine content increases with heating time and temperature, with time being more influential. And 23 out of 54 lysines in lactotransferrin were lactosylated. Following in-vitro infant digestion, free lysine levels in samples heated at 130 °C for 30s decreased by 25 % compared to unheated ones, likely due to lactosylation hindering protease cleavage. Intriguingly, lactosylated lysine was absent in peptides ranging from 5 to 17 amino acids but remained in larger peptides. The formation of large lactosylated peptides from heating impeded free lysine release. Further investigation is needed to determine if the human body can utilize these lactosylated peptides.

Contact dermatitis secondary to povidone-iodine: A systematic review.

Cutaneous reactions to povidone (PVP)-iodine are widely reported; however, distinction between allergic and irritant reactions can be challenging. Free iodine is responsible for irritant reactions and is released when PVP-iodine is in a liquid state. The aim of this study was to review the clinical presentation and results of patch testing in patients with PVP-iodine contact dermatitis. A systematic review was conducted by searching Pubmed, MEDLINE, and Google Scholar databases for reports of contact dermatitis secondary to PVP-iodine. Data were collated including study design, patient age and gender identity, iodine exposure, skin biopsy findings, and patch test methodology and results. The search revealed 187 reports with 38 eligible studies; 30 case reports/case series and 8 retrospective cohort studies. Overall, there were 223 patients with PVP-iodine contact dermatitis. The commonest reaction was irritant contact dermatitis (51%), followed by allergic contact dermatitis (40%) and contact dermatitis not further specified (9%). Irritant reactions were characterised by burn-like morphology and, when due to surgical skin disinfectant, were often distant from the surgical incision site. Patch testing was most often performed with a 10% PVP-iodine aqueous solution; however, irritant reactions in controls occur. Various testing methods including iodine in petrolatum, ethanol, dried powder, and open application testing were described. Most reactions to PVP-iodine are irritant and patch testing using a closed-chamber method yields inconsistent results due to risk of irritation from free iodine release over the 2-day occlusion time. Surgeons should be aware of the risk of prolonged skin contact with wet iodine solution.

Purple Butter Clam (Saxidomus Purpurata) as a Potential Functional Food Source for Obesity Treatment.

Saxidomus purpurata extract (SPE) is a highly consumable seafood worldwide with known health-related benefits. However, there are no reports of its' anti-obesity effect. This study explores the potential of SPE for anti-obesity effects by modulating adipogenesis and lipolysis. SPE reduced intracellular lipid and triglyceride accumulation while increasing free glycerol release in adipocytes. SPE inhibited lipogenesis protein expressions and increased the phosphorylation of hormone-sensitive lipase and Adenosine monophosphate-activated protein kinase (AMPK) to promote lipolysis. In addition, SPE suppressed adipogenesis by downregulating protein expression of key adipogenic markers, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) via Wnt/β-catenin signaling. SPE augmented the heme oxygenase-1 (HO-1) expression. Thus, pharmacological intervention with Zinc protoporphyrin (ZnPP-HO-1 antagonist) was employed to validate the HO-1 role. The presence of ZnPP increased the lipid accumulation and reduced the free glycerol release. At the molecular level, adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) expressions were restored in the presence of ZnPP. GC-MS analysis revealed that SPE was comprised of several fatty acids, contributing to its anti-obesity activity. SPE is an effective nutraceutical that can be used to reduce the progression of obesity. HO-1 expression during adipogenesis might be the mechanism of action for the anti-obesity effect of SPE.

DNA demethylation triggers cell free DNA release in colorectal cancer cells

BackgroundLiquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release.MethodsWe measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release.ResultsHigher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding.ConclusionsMethylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

7990 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background: Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods: 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results: AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion: Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

9225 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background Corticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution & ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p<0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

Chronic microcystin-leucine-arginine exposure induces osteoporosis by breaking the balance of osteoblasts and osteoclasts

Microcystin-leucine-arginine (MC-LR) produced by cyanobacterial harmful algal blooms are hazardous materials. However, the toxicity and mechanisms of continuous exposure to MC-LR on the occurrence of osteoporosis remains poorly documented. In this study, to mimic the chronic influences of MC-LR on the bone tissues in humans, an animal model was constructed in which mice were treated with MC-LR through drinking water at an environmentally relevant level (1–30 μg/L) for 6 months. MC-LR was enriched in the skeletal system, leading to the destruction of bone microstructure, the decrease of bone trabecular number, the reduction of osteoblasts, the enhanced content of lipid droplets, and the activation of osteoclasts, which is the characteristic of osteoporosis. Herein, we revealed ferroptosis is a vital mechanism of osteoblast death in mouse models of MC-LR. MC-LR exposure activates AMPK/ULK1 signaling, further promotes ferritin selective autophagy, causes free iron release and lipid peroxidation deposition, and eventually leads to ferroptosis of osteoblasts. Importantly, the use of AMPK or ferroptosis inhibitors in vivo markedly reduced MC-LR-induced osteoblast death and impaired osteogenic differentiation. Interestingly, MC-LR exposure promotes iron uptake in bone marrow macrophages through the TF-TFR1 pathway, leading to its transformation to TRAP-positive pre-osteoclast cells, thereby promoting bone resorption. Overall, our data innovatively revealed the core mechanism of MC-LR-induced osteoporosis, providing the bi-directional regulation of MC-LR on osteoblast-osteoclast from the perspective of iron homeostasis imbalance.

Edible Vitalmelon Fruit Extract Promotes Lipolysis of MDI-stimulated 3T3-L1 Adipocytes Through the Stimulation of Glycerol Release and β-oxidation

Background and Purpose: The anti-obesity effects of vitalmelon extracts have been well verified in only adipogenesis and lipogenesis of adipocytes and high-fat diet-induced obesity mice except lipolysis. Therefore, we were investigated the novel function of edible vitalmelon fruit water extract (VW) on lipolysis. Methods: The changes in the free glycerol release, cyclic adenosine monophosphate (cAMP)-dependent signaling pathway, and expression of β-oxidation-related regulators were analyzed in 3‐isobutyl‐1‐methylxanthine, dexamethasone, and insulin (MDI)-stimulated 3T3‐L1 adipocytes (MiSt-3T3 adipocytes) after a VW treatment. Results: The successful differentiation of pre-adipocytes was confirmed with increased oil-red O (ORO)-stained lipid accumulation and PPARγ gene transcription. After maturation of adipocytes, the levels of free glycerol, intracellular cAMP, and hormone-sensitive lipase phosphorylation increased in a dose-dependent manner in the MDI+VW-treated group. In addition, the protein and mRNA levels of three β-oxidation-related genes (ACADs, ACO1, and CTP) were remarkably increased in the MiSt-3T3 adipocytes after VW treatment, while ATPCL phosphorylation was decreased. The changes in the expression levels of these proteins were accompanied by alteration on the expression levels of the PPARα transcription factors. The levels of PPARα protein phosphorylation and mRNA transcription in MiSt-3T3 adipocytes increased dose-dependently after the VW treatment. Conclusion: These results show that VW can promote lipolysis by controlling the cAMP-mediated glycerol release pathway and β-oxidation in MiSt-3T3 adipocytes.

Effect of the whey protein isolate-pectin bilayer emulsion system on the textural properties and oil release characteristics of room-temperature stored processed cheese

Processed cheeses stored at room temperature exist some texture defects, such as texture softening and free oil release. The emulsion obtained by pre-emulsifying butter with whey protein isolate (WPI) can significantly improve the texture softening, but it was unable to mitigate the release of free oil during storage. Therefore, in this study, a WPI-pectin bilayer emulsion system was manufactured and applied in the production of processed cheese. The effects of adding the WPI-pectin bilayer emulsion system on the texture and functional characteristics of processed cheese during storage were investigated. The results of the study showed that the WPI-pectin bilayer emulsion system significantly improved the texture softening and free oil release characteristics of the room-temperature stored (RTS) processed cheese. Scanning electron microscopy revealed that the addition of the WPI-pectin bilayer emulsion system increased the uniformity of cheese microstructure and the number of small free fat pools after pre-emulsification. This study provided a method for improving the texture defects of RTS processed cheese, which is important for the processing and promotion of RTS processed cheese.

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Improving the physicochemical and sensory properties and volatile profile of goat ricotta cream with Limosilactobacillus mucosae CNPC007 supplementation

This study assessed the effects of the autochthonous strain Limosilactobacillus mucosae CNPC007 on the techno-functional characteristics, physicochemical properties, volatile profile, and sensory aspects of goat ricotta cream during refrigerated storage. Three cheese formulations were tested: CRC (without probiotic), RCLM (with L. mucosae CNPC007), and RCLA (with commercial probiotic Lactobacillus acidophilus La-5). L. mucosae CNPC007 influenced color, adhesiveness, lactose content, acidity, and proteolysis depth index in goat ricotta cream. RCLA and RCLM maintained a probiotic viable cell count >6 log CFU g−1. RCLM had the highest proteolysis depth index and free amino acid release. Seventeen distinct volatile compounds were identified in the formulations. Probiotic supplementation did not affect global acceptance or purchase intention. RCLA and RCLM were preferred over CRC in the ranking test and considered ideal for goat aroma in the JAR test. L. mucosae CNPC007 supplementation has the potential to improve the nutritional, functional, aromatic, and sensory aspects of goat ricotta cream.

The effect of carbetocin on the mammary gland of pigs

Carbetocin (deamino-1-monocarba-(2-Omethylthyrosine)-oxytocin) refers to a prolonged-acting, synthetic analogue of oxytocin, has pharmacodynamic properties similar to natural oxytocin. Drugs containing carbetocin have been used in the pathology of childbirth and the postpartum period. However, there is limited information in the literature on the effect of this pharmacological agent on the mammary gland of animals. The aim of the presented experimental work was to study the effect of carbetocin on the mammary gland of sows in the postpartum period. The studies were performed on the main sows in the early postpartum period. According to the principle of analogues, the animals were divided into 2 groups: experimental and control. Carbetocin was parenterally administered to sows of the experimental group at a dose of 0.07 mg with an interval of 24 hours, and uterotonic agents were not used in the control group. In animals of the experimental group, the time of induced secretion of breast secretions after administration of cabetocin was recorded. The concentration of immunoglobulins in colostrum (milk) of all studied animals was determined using a Brix refractometer. It was found that after carbetocin injection, the average duration of excretion of mammary gland secretions in sows had no significant differences during 1 and 2 days after delivery and amounted to 363.00±8.40 and 355.50±8.51 minutes, respectively. Its free release in animals occurs within 4-7 hours after administration of a prolonged oxytocin analog. The use of carbetocin in sows in the postpartum period does not lead to an increase in the amount of immunoglobulins in colostrum or transitional milk, regardless of the proportion of the mammary gland. At the same time, a significant decrease in immune proteins is recorded after 24 hours to the initial level.

Abstract 1871: Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity

Abstract EGFR and cMET are proven cancer targets co-expressed in diverse tumor types. EGFR × cMET bispecific antibody has been approved for the treatment of NSCLC, supporting a simultaneous targeting strategy. In addition to this dual targeting benefit, bispecific antibody drug conjugates (ADCs) targeting EGFR and cMET have also been developed to further improve anti-tumor activity and tissue selectivity. Sufficient target affinity, good cellular internalization and high tumor infiltration are critical for an ADC to mediate therapeutic activity. To this end, we developed the first EGFR × cMET bispecific nanobody conjugated with monomethyl auristatin E (MMAE) as payload (NXV01c). Lead nanobodies against EGFR and cMET were respectively selected from immune libraries by phage display, followed by highly efficient humanization and optimization by neoX’s computation platform. The bispecific nanobody with Fc (NXV01) was constructed by “knobs-into-holes” heterodimerization and then homogeneously conjugated with MMAE via a lysosomal cleavable valine-citrulline dipeptide linker. The resulting bispecific nanobody drug conjugate (NDC), NXV01c, was evaluated in multiple tumor cell lines and tumor xenograft models. NXV01, the pre-conjugate bispecific nanobody, bound EGFR and cMET with nanomolar potency (BLI) and inhibited the phosphorylation of cellular EGFR and cMET with nanomolar IC50 (ELISA). Moreover, it did not activate cellular cMET. Importantly, the NDC NXV01c is highly homogeneous: it has an average DAR of 3.87 and >95% of NXV01c has a DAR of 4. To examine stability of conjugation, NXV01C was incubated in human plasma (37°C) for 14 days, the maximum free drug release rate (by LC/MS/MS) is 0.68%, which is much lower than that of DS8201. In vitro, NXV01c was rapidly internalized into H1975 cells which co-expressed EGFR and cMET. It inhibited the growth of H1975 (lung) and SNU5 (gastric) cancer cells with picomolar activity but spared normal keratinocytes. NXV01c also inhibited the proliferation of additional cell lines derived from lung, gastric, esophageal, and liver cancer, and the level of inhibition positively associated with the expression density of both targets. In an H1975 cell line-derived xenograft model, NXV01C exhibited potent and dose-dependent anti-tumor activity. Treatments at 3 and 10 mg/kg once per week for 2 weeks led to shrinkage and complete regression of tumor, respectively. In patient-derived xenograft models of NSCLC and esophageal cancer, NXV01C led to tumor regression and elimination without noticeable toxic effects. EGFR × cMET bispecific nanobody drug conjugate NXV01c has favorable drug-like properties and demonstrated superior anti-tumor effect in vitro and in vivo. The results suggest NXV01C can be an effective solution for EGFR/cMET bearing tumors commonly found in diverse malignancies. Citation Format: Ran Wu, Puwei Yuan, Yang Xie, Jianxiu Guo, Fei Zhang, Fan Liu, Taylor B. Guo. Discovery and characterization of NXV01c, an EGFR × cMET bispecific nanobody drug conjugate with potent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1871.

Abstract 2114: Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor

Abstract Background: EGFR tyrosine kinase inhibitors (TKIs) have significantly improved the survival rate and the quality of life of the NSCLC patients with EGFR mutation in the first line setting. For example, Osimertinib, a potent third-generation EGFR TKI, has demonstrated exceptional efficacy in metastatic NSCLC with specific EGFR mutations. Despite its success, the emergence of drug resistance, often linked to elevated HER3 protein expression, remains a significant challenge. U3-1402 (HER3-Dxd), an HER3 ADC, demonstrates a promising result in managing Osimertinib-resistant NSCLC. It has been reported that combining U3-1402 with Osimertinib has shown enhanced efficacy in resistant cell lines and mouse models. Here, we explore a novel HER3 dual-payload ADC (HER3 dpADC), uniquely combining a Topoisomerase I inhibitor and an EGFR TKI within a single antibody structure using the enzymatic site-specific conjugation platform developed by GeneQuantum (GQ). This innovative approach demonstrates potent and synergistic anti-tumor effects in vitro and in vivo, presenting an alternative therapeutic strategy for EGFR-mutant NSCLC patients. Results: GQ's dpADC platform technology employs two orthogonal enzymatic site-specific conjugations and stable linker technologies to efficiently generate the dual-payload ADC with high homogeneity and quality. For HER3 dpADC, HER3 Ab was conjugated with a novel Topoisomerase I inhibitor, TopoIx, and an EGFR tyrosine kinase inhibitor. In vitro DAR analysis revealed high linker stability, with minimal free payload release even after 96 hours of plasma incubation. Binding affinity assessments confirmed comparable affinity between HER3 dpADC and HER3 mAb in specific EGFR exon 19 deletion cell lines. Using various NSCLC cell lines in a 3D-spheroid culture system, HER3 dpADC demonstrated significant dose-dependent and synergistic anti-tumor activities. A robust bystander killing efficacy was observed in both HER3+/HER3- HEK293T cell coculture assays. Evaluation in CDX mouse models consistently demonstrated heightened in vivo efficacy by HER3 dpADC, aligning with the promising in vitro data. In NSCLC patient-derived xenograft (PDX) mouse models representing EGFR tyrosine kinase inhibitor-sensitive or resistant phenotypes, HER3 dpADC induced tumor regressions with no obvious toxicity, underscoring its potential as the next-generation HER3 targeting agent. Conclusion: The innovative HER3 dpADC, leveraging an efficient dual enzymatic site-specific conjugation, exhibited robust anti-tumor efficacy in both in vitro and in vivo settings, surpassing single-agent treatments. Its synergistic mechanism of action presents a promising possibility for developing a more potent and the front-line therapeutic solutions in NSCLC patients who have progressed on standard therapies. Citation Format: Lina Wang, Meijun Xiong, Xinju Gao, Chengang Zhou, Yu Han, Yanchun Li, Junhao Wang, Lili Shi, Gang Qin, Paul H. Song. Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2114.

A case report on diabetic ketoacidosis in 5 years old female child

Diabetic ketoacidosis is characterised by an insulin deficiency, metabolic acidosis and increased ketone concentration in the body which can be normalised by insulin replacement and electrolyte replacement therapy. One in three children suffers with type 1 diabetes (T1D), across the US and globally, the incidence of the disease is rising among the paediatrics by over 3% annually. The diagnosis of diabetic ketoacidosis (DKA) was based on the presence of hyperglycaemia (blood glucose > 11 mmol/L), acidosis (serum bicarbonate < 15 mmol/L) and ketonuria (urine ketone ≥1+). Gestational diabetes is a common abnormality of glucose metabolism during pregnancy which affects foetal development and an alteration in the balance of glucagon and insulin is affected due to an overproduction of glucose and ketones in the liver, with free release of fatty acids from adipose tissue. A case of 5 years old female child presented with increased urination, increased appetite and sudden weight loss since 1 month and her RBS range was found to be 426mg/dl. Child underwent laboratory investigations, GRBS monitoring every 3 and assessed as Diabetic ketoacidosis with uncontrolled sugars. The child was treated with insulin supplementation for 3 days, recovered and child was hemodynamically stable and got discharged.

Comparison of Milk Kefirs Obtained from Cow's, Ewe's and Goat's Milk: Antioxidant Role of Microbial-Derived Exopolysaccharides.

Different types of milk are used in the production of milk kefir, but little information is available on the release of potentially antioxidant exopolysaccharides (EPS). The aim of this study was to investigate whether the microbial dynamics and EPS release are dependent on the milk substrate. In our study, the inoculated microbial consortium was driven differently by each type of milk (cow, ewe, and goat). This was evident in the sugar consumption, organic acid production, free amino release, and EPS production. The amount and the composition of the secreted EPS varied depending on the milk type, with implications for the structure and functional properties of the EPS. The low EPS yield in ewe's milk was associated with a higher lactic acid production and thus with the use of carbon sources oriented towards energy production. Depending on the milk used as substrate, the EPS showed different monosaccharide and FT-IR profiles, microstructures, and surface morphologies. These differences affected the antiradical properties and reducing power of the EPS. In particular, EPS extracted from cow's milk had a higher antioxidant activity than other milk types, and the antioxidant activity was concentration dependent.

Development and Characterization of Bio-Based Formaldehyde Free Sucrose-Based Adhesive for Fabrication of Plywood.

In order to solve the problem of excessive consumption of petrochemical resources and the harm of free formaldehyde release to human health, biomass raw materials, such as sucrose (S) and ammonium dihydrogen phosphate (ADP) can be chemically condensed in a simple route under acidic conditions to produce a formaldehyde free wood adhesive (S-ADP), characterized by good storage stability and water resistance, and higher wet shear strength with respect to petroleum based phenolic resin adhesive. The dry and boiling shear strength of the plywood based on S-ADP adhesive are as high as 1.05 MPa and 1.19 MPa, respectively. Moreover, is Modulus of Elasticity (MOE) is as high as 4910 MPa. Interestingly, the plywood based on the developed S-ADP adhesive exhibited good flame retardancy. After burning for 90 s, its shape remains unchanged. Meanwhile, it can be concluded from thermomechanical analysis (TMA) and thermogravimetric analysis (TGA) that the S-ADP acquired excellent modulus of elasticity (MOE) and good thermal stability. It is thus thought promisingly that the use of S-ADP adhesive as a substitute for PF resin adhesive seems feasible in the near future.

Metastability, adiabatic shear bands initiation and plastic strain localization in the AMg6 alloy under dynamic loading

New conception of adiabatic shear bands (ASB) and adiabatic shear failure mechanisms are proposed as special type of critical phenomen, structural-scaling transition, in the ensembles of microshears, governed by the characteristic non-linearity (metastability) of stored (free) energy of solid with mesodefects. Corresponding free energy release kinetics provides experimentally observed ASB induced staging of plastic strain localization and transition to adiabatic shear failure. ASB staging follows to collective properties of microshears ensemble given by the self-similar solutions of evolution equation providing spatial-temporal microshears localization, momentum transfer and damage localization. The criticality of ASB induced plastic strain localization and failure allows us to avoid the discrepancy in the interpretation of ASB effects as thermo-plastic instability in the balance of the stored energy and structural DRX transformation. The microshear ensemble is considered as the second phase and initiation of collective modes provide different staging according to the metastability decomposition and ASB scaling properties following to the self-similar solutions. Self-similar nature of microshears collective modes providing the ASB dynamics is analyzed as the mechanism of steady plastic wave front unversality in shocked materials. The dynamic split Hopkinson pressure bar tests were conducted with AlMg6 alloy combined with “in-situ” imaging of temperature kinetics by CEDIP Silver 450M high-speed infrared camera with conclusion of the secondary role of thermoplastic instability at the ASB staging. The microstructural study performed by an electron microscopy revealed the correlated behavior of the ensemble of defects, which can be classified as a structural transition and precursor of ASB induced strain localization and failure. The modeling reflecting the links of self-similar solutions in microshear ensembles with relaxation properies and damage localization was applied for the comparative analysis of ASB staging and temperature dynamics given be the infrared imaging.