Fragmentation Chain Research Articles

Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.

Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.

Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.

Heme-catalyzed degradation of linoleate 9-hydroperoxide (9-HPODE) forms two allylic epoxy-ketones via a proposed pseudo-symmetrical diepoxy radical intermediate

Heme-initiated decomposition of unsaturated fatty acid hydroperoxides creates alkoxyl radicals that propagate a complex series of reactions to hydroxy, keto, epoxy and aldehydic products. Herein, among the products from the hematin-catalyzed degradation of 9-hydroperoxy-linoleic acid (9-HPODE), we observed a double peak on normal-phase HPLC that resolved on RP-HPLC into equal proportions of two epoxy-allylic ketones with identical UV spectra. Their proton NMR spectra were also indistinguishable and consistent with 9,10-trans-epoxy-11E-13-keto- and 9-keto-10E-12,13-trans-epoxy-octadecenoic acids. Acid hydrolysis to the corresponding dihydroxy-ketones and GC-MS analysis identified the earlier eluting product on RP-HPLC as the 9,10-epoxy regio-isomer. Starting from the C9-hydroperoxide, recovery of the two epoxy-ketones in equal proportions suggests their formation from a common intermediate. Earlier work has proposed formation of a pseudo-symmetrical diepoxy radical (9,10-epoxy-11(•)-12,13-epoxy, derived from an epoxy allylic hydroperoxide precursor) in the carbon chain fragmentation leading to aldehydic products. This intermediate in pathways of alkoxyl radical reactions forms equal pairs of aldehydes, and now also a pair of epoxy-ketones, and based on mechanism the same products arise from either 9-HPODE or 13-HPODE. Our results point to the intermediacy of this diepoxy-carbinyl radical in the origin of at least two classes of linoleate peroxidation products, and it should be considered as a viable intermediate for homo-conjugated diene peroxidation in general. The reactions could contribute to the aldehydes and epoxy-ketones in tissues undergoing oxidative transformations of polyunsaturated fatty acids.

Mesoscale modeling of random chain scission in polyethylene melts

Polyolefins account for more than half of global primary polymer production, however only a small fraction of these polymers are currently being recycled. Fragmentation of polymer chains into shorter chains with a targeted molecular weight distribution with the goal of reusing these fragments in subsequent chemical synthesis can potentially introduce an alternative approach to polyolefins recycling. Herein we develop a mesoscale framework to model degradation of polyethylene melts at a range of high temperatures. We use the dissipative particle dynamics approach with modified segmental repulsive potential to model the process of random scission in melts of linear polymer chains. We characterize the fragmentation process by tracking the time evolution of the distribution of degrees of polymerization of chain fragments. Specifically, we track the weight average and the number average degrees of polymerization and dispersity of polymer fragments as a function of the fraction of bonds broken. Furthermore, we track the number fraction distribution and the weight fraction distribution of polymer fragments with various degrees of polymerization as functions of the fraction of bonds broken for a range of high temperatures. Our results allow one to quantify to what extent the distribution of polymer chain fragments during random scission can be captured by the respective analytical distributions for the range of conversions considered. Understanding the thermal degradation of polyolefins on the mesoscale can result in the development of alternative strategies for recycling a range of thermoplastics.

Amyloid Neuropathy: From Pathophysiology to Treatment in Light-Chain Amyloidosis and Hereditary Transthyretin Amyloidosis.

Amyloid neuropathy is caused by deposition of insoluble β-pleated amyloid sheets in the peripheral nervous system. It is most common in: (1) light-chain amyloidosis, a clonal non-proliferative plasma cell disorder in which fragments of immunoglobulin, light or heavy chain, deposit in tissues, and (2) hereditary transthyretin (ATTRv) amyloidosis, a disorder caused by autosomal dominant mutations in the TTR gene resulting in mutated protein that has a higher tendency to misfold. Amyloid fibrils deposit in the endoneurium of peripheral nerves, often extensive in the dorsal root ganglia and sympathetic ganglia, leading to atrophy of Schwann cells in proximity to amyloid fibrils and blood-nerve barrier disruption. Clinically, amyloid neuropathy is manifested as a length-dependent sensory predominant neuropathy associated with generalized autonomic failure. Small unmyelinated nerves are involved early and prominently in early-onset Val30Met ATTRv, whereas other ATTRv and light-chain amyloidosis often present with large- and small-fiber involvement. Nerve conduction studies, quantitative sudomotor axon testing, and intraepidermal nerve fiber density are useful tools to evaluate denervation. Amyloid deposition can be demonstrated by tissue biopsy of the affected organ or surrogate site, as well as bone-avid radiotracer cardiac imaging. Treatment of light-chain amyloidosis has been revolutionized by monoclonal antibodies and stem cell transplantation with improved 5-year survival up to 77%. Novel gene therapy and transthyretin stabilizers have revolutionized treatment of ATTRv, improving the course of neuropathy (less change in the modified Neuropathy Impairment Score + 7 from baseline) and quality of life. With great progress in amyloidosis therapies, early diagnosis and presymptomatic testing for ATTRv family members has become paramount. ANN NEUROL 2024;96:423-440.

Thermal decomposition mechanism of HMX/HTPB hybrid explosives studied by reactive molecular dynamics.

The thermal decomposition process of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine/hydroxyl-terminated polybutadiene (HMX/HTPB) hybrid explosives and pure HMX explosives at different temperatures (2000 ~ 3500 K) was investigated using the reactive molecular dynamics method. This study aimed to analyze the effect of binders on the thermal decomposition of HMX at the atomic scale and reveal the thermal decomposition mechanism of HMX/HTPB. The results showed that the thermal decomposition process of the HMX molecule in the HMX/HTPB hybrid system involves a continuous denitration followed by the disintegration of the main ring. The HTPB chain will experience dehydrogenation, dehydroxylation, and chain fragmentation. Including HTPB in the hybrid system significantly increased the presence of H and OH radicals. These radicals then interacted with HMX and its decomposition products and produced more of the final products H2O and H2 in the HMX/HTPB hybrid system compared to pure HMX. Additionally, it was observed that the HTPB chain fragments attached to the HMX decomposition products prevented the formation of N2 and CO2. Furthermore, the activation energies (Ea) of the initial and intermediate decomposition stages of the HMX/HTPB hybrid system were 98.45 kJmol-1 and 90.69 kJmol-1, respectively. The results showed that the activation energies of the HMX/HTPB hybrid system are lower than the pure HMX system in these two stages. Consequently, HTPB will enhance HMX's thermal decomposition and decreased the system's insensitivity to heat stimuli. The molecular dynamics simulation of the HMX/HTPB hybrid system was performed using the ReaxFF module in the LAMMPS software, and the ReaxFF/lg force field was used to describe the interatomic interactions as well as the chemical reactions.

Structural characterization and innate immunomodulatory effect of glucomannan from Bletilla striata

The crude polysaccharide of Bletilla striata in this study was extracted by water extraction and alcohol precipitation and further purified by gel column to yield the purified component Bletilla striata polysaccharide (BSP). Its structure and innate immune regulation activity were studied. BSP mainly comprises mannose and glucose, with a monosaccharide molar ratio of 2.9:1 and a weight-average molecular weight of 28,365 Da. It is a new low-molecular-weight water-soluble neutral glucomannan. BSP contains a → 6)-β-Manp-(1→, →4)-β-Glcp-(1→, →4)-β-Manp-(1 → and →3)-α-Manp-(1 → linear main chain, containing β-Glcp-(1 → and β-Manp-(1 → two branched chain fragments were connected to the Man residue at position 4. BSP can enhance the anti-infection ability of Caenorhabditis elegans against Pseudomonas aeruginosa, significantly improve the phagocytic ability of RAW264.7 macrophages, stimulate the secretion of NO and TNF-α, and have good innate immune regulation activity. These findings guide the use of Bletilla striata polysaccharides with immunomodulatory action.

Cyclic Silicon Organic Copolymers: Synthesis and Investigation. Review

This paper considers the synthesis and investigation of cyclic silicon-organic polymers with mono- and polycyclic fragments in the side chain. For obtaining monocyclic polymers, the hydrosilylation reaction of 1-hydro-3-vinylhexamethylcyclotetrasiloxane was used. The reaction was conducted in a CCl4 solution at 75°C in the presence of Speier’s catalyst (H2PtCl6  6H2O) to produce a viscous-flow at room temperature polymer. The polymers were studied by NMR spectroscopy. Poly(carbosiloxane) with cyclic fragments in the methyl-siloxane backbone was synthesized by the hydride polyaddition of divinylorganocyclosiloxane with dihydrodimethylsiloxane. A semi-quantitative assessment conducted using NMR spectroscopy revealed the ratio of isomeric 1,3- and 1,5-cyclic structures as 1:1. X-ray diffraction studies indicated that copolymers are single-phase amorphous systems. Also, in the review, synthesis and studies of carbosiloxane copolymers containing flexible dimethylsiloxane and decaorganotricyclodecasiloxane fragments in the backbone are discussed. Hydride polyaddition of divinyl-containing compounds was carried out for -dihydridedimethylsiloxanes of various lengths. The synthesized copolymers were characterized by the X-ray diffraction method and TGA.

CRPA1A2: A novel TCR-based T-cell engager targeting MAGEA1-positive solid tumors.

e14589 Background: MAGEA1, identified as a cancer/testis antigen, is highly expression in various solid tumors, including hepatocellular carcinoma, non-small cell lung cancer, but absent in normal tissues except male germ cells and placenta, both of which lacking HLA class molecules. This selective expression of HLA-MAGEA1 epitope makes it a promising target for cancer therapy. However, its intracellular localization poses a challenge for conventional antibodies designed to target surface-expressed antigens. TCR-based T-cell engagers, such as Tebentafusp, overcome this hurdle by targeting intracellular protein-derived peptides presented on cancer cell surfaces via HLA, enabling precise cytotoxic T cell attacks. Notably, Tebentafusp has shown significant clinical benefits by improving survival rates in metastatic uveal melanoma, underscoring the efficacy of TCR-based T-cell engagers in treating MAGEA1-positive tumors. Methods: We have developed an advanced structural format, CR62, consisting of a anti-CD3 single chain fragment, a soluble modified TCR, and an Fc region. Additionally, the use of CorreGene's SMART-TCR system has facilitated the acquisition of TCRs with high affinities in the pico-molar range. By leveraging CR62 and a high-affinity TCR, we have conceptualized the TCR-based T-cell engager, CRPA1A2. Comprehensive preclinical assessments have been conducted to evaluate CRPA1A2's safety and efficacy. Results: CRPA1A2 is designed to specifically target the HLA-A*02:01-restricted MAGEA1 epitope, effectively inhibiting tumor growth. CRPA1A2 is characterized by its exceptional soluble expression in CHO cells, minimal homodimer miss-assembly and simplifying purification. It is strategically engineered with a high-affinity TCR and a low-affinity anti-CD3 scFv to optimize the efficacy and safety. In vitro studies, CRPA1A2 showed strong ability to eliminate tumor cells and induce IFN-γ release, effective even at low MAGEA1 epitope densities, with a therapeutic window that spans over three orders of magnitude, suggesting a 1000-fold greater activity against antigen-positive cancer cells compared to antigen-negative cells. In vivo, using a tumor-bearing mouse model, CRPA1A2 has been shown to recruit T cells into subcutaneous solid tumors, expand CD8+ T cell populations, and significantly reduce tumor mass. Notably, CRPA1A2 has an extended half-life of 120 hours and demonstrates preferential accumulation in tumor tissues, thereby enhancing its therapeutic potential. Moreover, the safety assessments for CRPA1A2 highlight its exceptional specificity, with in vitro studies showing no evidence of off-target toxicity, on-target off-tumor toxicity, or alloreactivity and favorable tolerance in mice during in vivo studies. Conclusions: CRPA1A2 has emerged as a superior TCR-based T-cell engager with exceptional antitumor efficacy and promising safety profile.

Dose-dependent effects of gamma radiation sterilization on the collagen matrix of human cortical bone allograft and its influence on fatigue crack propagation resistance

Fatigue crack propagation resistance and high-cycle S–N fatigue life of cortical bone allograft tissue are both negatively impacted in a radiation dose-dependent manner from 0 to 25 kGy. The standard radiation sterilization dose of 25–35 kGy has been shown to induce cleavage of collagen molecules into smaller peptides and accumulation of stable crosslinks within the collagen matrix, suggesting that these mechanisms may influence radiation-induced losses in cyclic fracture resistance. The objective of this study was to determine the radiation dose-dependency of collagen chain fragmentation and crosslink accumulation within the dose range of 0–25 kGy. Previously, cortical bone compact tension specimens from two donor femoral pairs were divided into four treatment groups (0 kGy, 10 kGy, 17.5 kGy, and 25 kGy) and underwent cyclic loading fatigue crack propagation testing. Following fatigue testing, collagen was isolated from one compact tension specimen in each treatment group from both donors. Radiation-induced collagen chain fragmentation was assessed using SDS-PAGE (n = 5), and accumulation of pentosidine, pyridinoline, and non-specific advanced glycation end products were assessed using a fluorometric assay (n = 4). Collagen chain fragmentation increased progressively in a dose-dependent manner (p < 0.001). Crosslink accumulation at all radiation dose levels increased relative to the 0 kGy control but did not demonstrate dose-dependency (p < 0.001). Taken together with our previous findings on fatigue crack propagation behavior, these data suggest that while collagen crosslink accumulation may contribute to reduced notched fatigue behavior with irradiation, dose-dependent losses in fatigue crack propagation resistance are mainly influenced by radiation-induced chain fragmentation.

Chain branching during the polycondensation of isophthalic aldehyde and 1,1′‐diacetylferrocene by the Claisen–Schmidt reaction

AbstractN‐methyl‐2‐pyrrolidone‐soluble ferrocene‐containing polychalcones, which are promising as a platform for the formation of ferromagnetic coatings after pyrolysis in an inert atmosphere is synthesized through the polycondensation of isophthalic aldehyde and 1,1′‐diacetylferrocene by the Claisen–Schmidt reaction. The structure and the molecular weight distribution of the polychalcones is characterized by NMR, IR spectroscopy, and size exclusion chromatography. It is shown that the formation of polychalcones is accompanied by chain branching via the Michael reactions and the aldol‐croton condensation. The structure of the linear fragments of the polychalcone chains is simulated with a tailored low‐molecular model compound, that is 1,3‐bis(3‐ferrocenyl‐1‐propen‐2‐one)benzene, which is characterized by the NMR and IR spectroscopy as well as the X‐ray diffraction analysis. The obtained results indicate an increasing tendency for polychalcone chain branching with the temperature growth during the Claisen–Schmidt reaction. The weight loss and the increase in magnetization under the conditions of pyrolysis of polychalcones at different temperatures is studied. The assumptions regarding the possible mechanisms of thermal destruction of ferrocene‐containing macromolecules are put forward.

Prognostic value of BRCA1 promoter methylation for patients with epithelial ovarian cancer

ObjectiveBRCA1 promoter methylation (BRCA1pm) is suspected to alter prognosis of patients with epithelial ovarian cancer (EOC). We aimed to evaluate the prognostic impact of this epigenetic modification. MethodsWe conducted a retrospective, monocentric study from 11/2006 to 08/2018. Patients with EOC and available status concerning somatic BRCA1/2 mutation and BRCA1pm were included. Three groups were defined: patients without BRCA1/2 mutation or BRCA1pm, patients with BRCA1/2 mutation and patients with BRCA1pm. BRCA1/2 mutations were analyzed in current care settings by next-generation sequencing (NGS). BRCA1pm analysis was assessed and quantified from bisulfite converted DNAs using fluorescent methylation specific polymerase chain reaction (PCR) and fragment analysis. All patients signed a consent form and the study was authorized by a Personal Protection Committee. Descriptive statistics were used to describe groups. Multivariate analysis was performed using the logistic regression model and including the variables that could be known at the time of diagnosis and that were significant at univariate analysis. Survival was compared between the groups. Kaplan-Mayer curves were used to express the differences in survival that were compared using log rank tests. Results145 patients were included: 95 (65.5 %) patients without BRCA1/2 mutation or BRCA1pm, 32 (22.1 %) patients with BRCA1/2 mutation, 18 (12.4 %) patients with BRCA1pm. Median survival was decreased in patients with BRCA1pm. Comparison of survival revealed a significant difference in overall survival (p = 0.0078) with a worse prognosis for patients with a BRCA1pm. ConclusionBRCA1pm in patients with EOC is an independent factor associated with a decreased overall survival. SynopsisBRCA1 promotor methylation in patients with epithelial ovarian cancer is an independent factor associated with a decreased overall survival.

SYNTHESIS OF POLYACRYLAMIDE HYDROGELS WITH PORPHYRIN FRAGMENTS IN THE SIDE CHAIN IN THE PRESENCE OF IMIDAZOLIUM IONIC LIQUIDS

The acrylamide hydrogels containing the porphyrin fragments have been obtained by radical polymerization in a solution. The synthesis has been carried out in the presence of imidazolium ionic liquids with length of the alkyl substituent in the imidazolium ring from C4 to C8. The influence of the used ionic liquids on the structure and some properties of the resulting porphyrin-containing acrylamide hydrogels has been established. The addition of an ionic liquid with a different length of the alkyl substituent into the reaction system can influence the structural changes that occur with the porphyrin comonomer during copolymerization with acrylamide. The formation of bacteriochlorin-like structures is inhibited in the presence of an ionic liquid in the reaction medium. This effect is most noticeable when using an imidazolium ionic liquid with a C8 alkyl substituent length. Hydrogels synthesized in the presence of ionic liquids have lower specific surface area compared to ones obtained without the use of ionic liquids. The lowest specific surface area values were shown by hydrogels synthesized using 1-octyl-3-methylimidazolium bromide. It was also found that the introduction of ionic liquid into the reaction mixture can contribute to both increase and decrease in the numerical values of the sorption characteristics of the resulting hydrogels, depending on the length of the alkyl substituent in the imidazolium ring and a porphyrin : acrylamide ratio. Hydrogels synthesized using 1-hexyl-3-methylimidazolium bromide and 1-octyl-3-methylimidazolium bromide at an initial porphyrin : acrylamide ratio of 1:20 have the highest degree of swelling. Varying the ratio of the initial monomers and the structure of the imidazolium ionic liquid during the copolymerization process allows to control the physicochemical characteristics of the resulting porphyrin-containing hydrogels, which can be useful for solving various applied problems. For citation: Pechnikova N.L., Smirnov A.S., Lyubimtsev A.V., Aleksandriiskiy V.V., Ageeva T.A. Synthesis of polyacrylamide hydrogels with porphyrin fragments in the side chain in the presence of imidazolium ionic liquids. ChemChemTech [Izv. Vyssh. Uchebn. Zaved. Khim. Khim. Tekhnol.]. 2024. V. 67. N 6. P. 73-79. DOI: 10.6060/ivkkt.20246706.7053.

Abstract PO4-06-08: ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER

Abstract Triple negative breast cancer (TNBC) has the worst prognosis of BC subtypes, largely due to lack of approved targeted therapies. Chimeric antigen receptor (CAR) T cells have demonstrated great success in certain hematological malignancies; however, in the treatment of solid cancers, CAR T cells are faced with a variety of obstacles that limit their efficacy, including on-target/off-tumor toxicity. To minimize these toxicities, the ideal CAR T cell target must be overexpressed by the tumor and absent from vital organs. Cancer/germline (CG) antigens satisfy this need, as their expression is restricted to male or female germ cells. Using a bioinformatic pipeline to identify novel CG antigens expressed in TNBC and other solid cancers, we have identified zona pellucida glycoprotein 4 (ZP4) as a candidate CG antigen target for CAR T cell therapy. This protein is a component of the human zona pellucida matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. We have identified ZP4 expression in approximately 10% of TNBC cases from available RNAseq and proteomic databases and have confirmed ZP4 expression in several TNBC PDX models. The overall purpose of this study is to redirect T cells against ZP4 by engineering and validating a second generation ZP4-specific CAR. We have generated a library of monoclonal antibody (mAb) candidates against the extracellular portion of human ZP4. Antibody clones were screened, and their specificity validated via ELISA and flow cytometry, and the three strongest binders (z108, z128, and z164) were selected for further investigation. By IHC, we confirmed that the ZP4 mAb clones stained the area surrounding normal oocytes and did not show binding to any of the 23 normal tissues screened. The variable regions of the antibody heavy and light chains were sequenced using 5’RACE and used to assemble second generation CAR constructs composed of the variable heavy and light chain fragments separated by a linker, a CH2/CH3 hinge domain, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and the CD3z signaling domain. Activated T cells transduced with retroviral supernatant express the ZP4 CAR constructs at high levels. While all ZP4 CAR T cells demonstrate antigen-specific cytotoxic killing of ZP4+ target tumor cells in short-term in vitro assays, z128 ZP4 CAR T cells demonstrate superior persistence and efficacy in long-term repeat tumor challenge in vitro assays and orthotopic in vivo mouse models. In summary, ZP4 is a promising target for TNBC and we have successfully engineered ZP4 specific CAR T cells. ZP4 CAR T cells effectively target and kill ZP4+ cells in in vitro cytotoxicity assays but perform differently in repeat tumor challenge assays and in vivo. Future studies will elucidate the precise differences between our three candidate CAR constructs and further test the effector function, trafficking, proliferation, and persistence of z128 ZP4 CAR T cells in vivo using ZP4+ PDX models. This work has exciting potential to bring a novel CAR T cell therapy to patients with TNBC, without concerns for on-target off-tumor toxicity. Citation Format: Lauren Somes, Jonathan Lei, Paul Shafer, Xinpei Yi, Diego Chamorro, Emily Madaras, Michael Lewis, Bing Zhang, Valentina Hoyos. ZP4 AS A NOVEL TARGET FOR CAR T CELL THERAPY IN TRIPLE NEGATIVE BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-06-08.

Myofibrillar protein lipoxidation in fish induced by linoleic acid and 4-hydroxy-2-nonenal: Insights from LC-MS/MS analysis

The oxidation of fish lipids and proteins is interconnected. The LOX (lipoxygenase)-catalyzed LA (linoleic acid) oxidation system on MPs (myofibrillar proteins) was established in vitro, to investigate the impact of lipoxidation on the physicochemical properties of fish MPs. By detecting HNE (4-hydroxy-2-nonenal) concentration during LA oxidation, the HNE treatment system was established to investigate the role of HNE in this process. In addition, the site specificity of modification on MPs was detected utilizing LC-MS/MS. Both treatments could induce sidechain modification, increase particle size, and cause loss of nutritional value through the reduction in amino acid content of MPs. The HNE group is more likely to alter the MPs' surface hydrophobicity compared to the LA group. By increasing the exposure of modification sites in MPs, the HNE group has more types and number of modifications compared to the LA group. LA group mainly induced the modification of single oxygen addition on MPs instead, which accounted for over 50 % of all modifications. The LA group induced a more pronounced reduction in the solubility of MPs as compared to the HNE group. In conclusion, HNE binding had a high susceptibility to Lys on MPs. Protein aggregation, peptide chain fragmentation, and decreased solubility occurred in the LA group mainly induced by peroxide generated during lipid oxidation or the unreacted LA instead of HNE. This study fills in the mechanism of lipoxidation on protein oxidation in fish and sheds light on the HNE modification sites of MPs, paving the way for the development of oxidation control technology.

Effect of rolling reduction on microstructure, mechanical properties and fracture features of hot isostatically pressed 30CrMnSiNi2A low alloy ultrahigh strength steel

As a powder metallurgy (PM) defect, existence of prior particle boundaries (PPBs) often makes mechanical properties visibly deteriorated. Hot rolling has been found to be an effective method of destroying PPBs. In present study, five different rolling reductions are applied to explore the effects of rolling reduction on evolution of PPBs in PM 30CrMnSiNi2A steels. Microstructure, mechanical properties and fracture modes of five samples are systematically analyzed and compared. Increase of rolling reduction not only makes Al-rich PPBs refined but also makes continuous PPBs structure gradually transform to dispersed oxides and short chain fragments, which can be attributed to deformation of powder particles. Change on distribution of PPBs leads to the variation of fracture modes. A strength-ductility-toughness balance is achieved when the rolling reduction scales up to 60 %. Although increase of rolling reduction causes grain refinement, work hardening and improvement of PPBs, it promotes formation of edge cracks. Occurrence of edge cracks is strongly associated with PPBs inclusions and makes ductility and toughness deteriorated.

Analyzing A Planet That Crosses a Small Comet’s Fragment Chain

This study explores the novel concept of a planet intersecting the orbit of a small comet's fragment chain. Utilizing a simple multi-generational model of comet fragmentation, it analyzes the planet's interaction with the comet’s fragment chain and simulates probability distributions for both chain crossing and fragment capture. This model offers insights complementary to impact theories that seek to explain the Younger Dryas boundary. Moreover, the potential presence of cometary material in Earth's orbit due to crossing a comet’s fragment chain could profoundly influence climate patterns, with far-reaching implications for global agriculture and food production.

CARs derived from broadly neutralizing, human monoclonal antibodies identified by single B cell sorting target hepatitis B virus-positive cells.

To design new CARs targeting hepatitis B virus (HBV), we isolated human monoclonal antibodies recognizing the HBV envelope proteins from single B cells of a patient with a resolved infection. HBV-specific memory B cells were isolated by incubating peripheral blood mononuclear cells with biotinylated hepatitis B surface antigen (HBsAg), followed by single-cell flow cytometry-based sorting of live, CD19+ IgG+ HBsAg+ cells. Amplification and sequencing of immunoglobulin genes from single memory B cells identified variable heavy and light chain sequences. Corresponding immunoglobulin chains were cloned into IgG1 expression vectors and expressed in mammalian cells. Two antibodies named 4D06 and 4D08 were found to be highly specific for HBsAg, recognized a conformational and a linear epitope, respectively, and showed broad reactivity and neutralization capacity against all major HBV genotypes. 4D06 and 4D08 variable chain fragments were cloned into a 2nd generation CAR format with CD28 and CD3zeta intracellular signaling domains. The new CAR constructs displayed a high functional avidity when expressed on primary human T cells. CAR-grafted T cells proved to be polyfunctional regarding cytokine secretion and killed HBV-positive target cells. Interestingly, background activation of the 4D08-CAR recognizing a linear instead of a conformational epitope was consistently low. In a preclinical model of chronic HBV infection, murine T cells grafted with the 4D06 and the 4D08 CAR showed on target activity indicated by a transient increase in serum transaminases, and a lower number of HBV-positive hepatocytes in the mice treated. This study demonstrates an efficient and fast approach to identifying pathogen-specific monoclonal human antibodies from small donor cell numbers for the subsequent generation of new CARs.

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

Advancement in the development of single chain antibodies using phage display technology.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.