Fragmentation Chain Research Articles

Preparation and Performance of PBAT/PLA/CaCO3 Composites via Solid-State Shear Milling Technology

Replacing traditional disposable, non-biodegradable plastic packaging with biodegradable plastic packaging is one of the key approaches to address the issue of “white pollution”. PBAT/PLA/inorganic filler composites are widely utilized as a biodegradable material, commonly employed in the field of packaging films. However, the poor dispersion of inorganic fillers in the polymer matrix and the limited compatibility between PBAT and PLA have led to inferior mechanical properties and elevated costs. In this work, we propose a simple and effective strategy to improve the dispersion of nano-CaCO3 in a PBAT/PLA matrix through solid-state shear- milling (S3M) technology, combined with mechanochemical modification and in situ compatibilization to enhance the compatibility between PBAT and PLA. The impact of varying milling conditions on the structure and performance of the PBAT/PLA/CaCO3 composites was investigated. During the milling process, PBAT and PLA undergo partial molecular chain fragmentation, generating more active functional groups. In the presence of the chain extender ADR during melt blending, more branched PBAT-g-PLA is formed, thereby enhancing matrix compatibility. The results indicate that the choice of milling materials significantly affects the structure and properties of the composite. The film obtained by milling only PBAT and CaCO3 exhibited the best performance, with its longitudinal tensile strength and fracture elongation reaching 22 MPa and 437%, respectively. This film holds great potential for application in the field of green packaging.

Characterization of the thermomechanical and aging behaviour of PAEK and CF/PAEK composites under long-term high-temperatures

The growing use of poly(aryl ether ketone) (PAEK) polymers reinforced with carbon fiber (CF) aims to meet the increasing demand for thermoplastic composites in applications that require excellent mechanical properties and thermal stability at higher temperatures. This study investigated the physicochemical properties of PAEK and CF/PAEK composites under long-term high-temperature exposure (150 °C, 200 °C and 250 °C) for durations of 15, 30, and 45 days, aiming to elucidate the durability mechanisms and enhance understanding of their performance. Specifically, the changes in the flexural properties and dynamic thermo-mechanical properties of the materials were evaluated after long-term high-temperature treatment. The results indicated that both PAEK and CF/PAEK composites possess substantial potential for long-term high-temperature applications, with CF not significantly altering the fundamental high-temperature response of the PAEK matrix. Regarding durability mechanisms, at 150 °C, PAEK's amorphous region undergoes chain reorientation, while temperatures of 200 °C and 250 °C induce significant secondary crystallization, which significantly improved the high-temperature mechanical properties of these materials. Additionally, at temperatures up to 250 °C, shorter linear chain fragments form through oxidative scission in PAEK's amorphous molecular chains. This work not only enriches the existing data on the properties of PAEK and CF/PAEK composites after long-term high-temperature treatment, but also provides further insights into their durability mechanisms.

Thermolysis of Cs9MO4 (M = In, Sc): Synthesis, Crystal Structure, Chemical Bonding, and Reactivity of the Subvalent Oxidometalates Cs7MO4 (M = In, Sc).

The cesium-poor alkali-metal suboxidometalates Cs7MO4 (M = In, Sc) were prepared from the respective cesium-rich suboxidometalates Cs9MO4 by thermolysis in a dynamic vacuum at temperatures below 150 °C. They crystallize in a new structure type, comprising isolated tetrahedral oxidometalate anions [MO4]5- immersed in a matrix of metallic cesium atoms. This structural separation into alternating ionic and metallic building units is typical for subvalent compounds. Cs7InO4 and Cs7ScO4 are isotypic and form mixed crystals Cs7(In1-xScx)O4 with statistic distribution of In and Sc atoms. All compounds were characterized by single crystal and powder X-ray diffraction. Calculations of the electronic structure with DFT methods corroborate the coexistence of anionic entities within a metallic matrix and give evidence of the subvalent nature of cesium atoms. Overall metallic behavior is a consequence of the equal volume fractions of metallic and ionic regions, allowing for percolation of the conduction electrons throughout the crystal volume. Differential thermoanalysis was employed to gain insight into the chemical conversion processes during the thermolysis reaction. The thermolytic decomposition of Cs9MO4 is a multistep process with, in some instances, reversible amorphization and recrystallization steps. Finally, the formation of ionic compounds is observed. The full decomposition of Cs9ScO4 yields the two new oxidoscandates Cs14Sc4O13 and Cs17Sc5O15 with tetrahedral [ScO4] units connected to chain fragments and the indium compound yields cesium oxidoindates with low-condensed [InO4] building units. The cesium-poor suboxidometalates Cs7MO4 themselves can serve as starting materials for the synthesis of further, new suboxidometalates, as they show a unique reactivity toward metals.

Dual Sources of S2 Observed in Comet 67P: Insights from Comparing ROSINA Measurements and Laboratory Simulations

Hydrogen sulfide (H2S) is the fifth most abundant molecule observed in the coma of comet 67P/Churyumov–Gerasimenko (67P). Prior to its incorporation into cometary materials, H2S likely underwent ultraviolet (UV) radiation exposure, which is thought to initiate a complex sulfur chemistry. We present an investigation into the UV photochemistry of H2S ices using infrared, Raman, and mass spectrometry techniques. Our study reveals the production of complex sulfur allotropes ranging from S2 to S6, alongside polysulfanes (H2S n , n = 2–3). Temperature-programmed desorption measurements postirradiation of H2S exhibit two peaks for S2 molecules: a broad peak between 80 and 140 K and a distinct peak at ∼245 K. Notably, larger allotropes S3–S5 exclusively display the 245 K peak. Furthermore, ROSINA measurements of the S2/H2S ratio during dust impact events and previously reported S2/H2S ratios in the undisturbed coma are compared to our laboratory-determined S2/H2S values. This analysis identifies two distinct sources of S2: a volatile S2 potentially sublimated directly from the comet’s surface and a secondary source likely resulting from fragmentation of larger sulfur chains during dust impacts. We determined the ratio of produced S2 to the initial H2S for both the volatile component and the refractory component at 245 K with both measurements conducted at an irradiation incident fluence of 2.25 × 1017 photons cm−2. These laboratory-derived S2/H2S ratios exhibit concordance with ROSINA measurements. When extrapolated to incident fluences anticipated in molecular clouds, this photoprocessing mechanism offers a plausible explanation for the measured S2/H2S ratio in comet 67P.

Engineering and physicochemical characterization of a novel, stable, symmetric bispecific antibody with dual target-binding using a common light chain.

Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.

Skeletal muscle myosin heavy chain fragmentation as a potential marker of protein degradation in response to resistance training and disuse atrophy.

We examined how resistance exercise (RE), cycling exercise and disuse atrophy affect myosin heavy chain (MyHC) protein fragmentation. The 1boutRE study involved younger men (n=8; 5±2years of RE experience) performing a lower body RE bout with vastus lateralis (VL) biopsies being obtained prior to and acutely following exercise. With the 10weekRT study, VL biopsies were obtained in 36 younger adults before and 24h after their first/naïve RE bout. Participants also engaged in 10weeks of resistance training and donated VL biopsies before and 24h after their last RE bout. VL biopsies were also examined in an acute cycling study (n=7) and a study involving 2weeks of leg immobilization (n=20). In the 1boutRE study, fragmentation of all MyHC isoforms (MyHCTotal) increased 3h post-RE (∼200%, P=0.018) and returned to pre-exercise levels by 6h post-RE. Interestingly, a greater magnitude increase in MyHC type IIa versus I isoform fragmentation occurred 3h post-RE (8.6±6.3-fold vs. 2.1±0.7-fold, P=0.018). In 10weekRT participants, the first/naïve and last RE bouts increased MyHCTotal fragmentation 24h post-RE (+65% and +36%, P<0.001); however, the last RE bout response was attenuated compared to the first bout (P=0.045). Although cycling exercise did not alter MyHCTotal fragmentation, ∼8% VL atrophy with 2weeks of leg immobilization increased MyHCTotal fragmentation (∼108%, P<0.001). Mechanistic C2C12 myotube experiments indicated that MyHCTotal fragmentation is likely due to calpain proteases. In summary, RE and disuse atrophy increase MyHC protein fragmentation. Research into how ageing and disease-associated muscle atrophy affect these outcomes is needed. HIGHLIGHTS: What is the central question of this study? How different exercise stressors and disuse affect skeletal muscle myosin heavy chain fragmentation. What is the main finding and its importance? This investigation is the first to demonstrate that resistance exercise and disuse atrophy lead to skeletal muscle myosin heavy chain protein fragmentation in humans. Mechanistic in vitro experiments provide additional evidence that MyHC fragmentation occurs through calpain proteases.

Productive chemistry induced by mechanochemically generated macroradicals.

Large or repeated mechanical loads degrade polymeric materials by accelerating chain fragmentation. This mechanochemical backbone fracture usually occurs by homolysis of otherwise inert C-C, C-O and C-S bonds, generating highly reactive macroradicals. Because backbone fracture is detrimental on its own and the resulting macroradicals can initiate damaging reaction cascades, a major thrust in contemporary polymer mechanochemistry is to suppress it, usually by mechanochemical release of "hidden length" that dissipates local molecular strain. Here we summarize an emerging complementary strategy of channelling mechanochemically generated macroradicals in reaction cascades to form new load-bearing chemical bonds, which enables local self-healing or self-strengthening, and/or to generate mechanofluorescence, which could yield detailed quantitative molecular understanding of how material-failure-inducing macroscopic mechanical loads distribute across the network. We aim to identify generalizable lessons derivable from the reported implementations of this strategy and outline the key challenges in adapting it to diverse polymeric materials and loading scenarios.

Boosting the toolbox for live imaging of translation.

Live imaging of translation based on tag recognition by a single-chain antibody is a powerful technique to assess translation regulation in living cells. However, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system, especially in a multicellular organism. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different green fluorescent protein variants fused to the single-chain fragment variable (scFv) and uncovered photobleaching, aggregation, and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

Aromatic polyamides with triarylmethane fragments in the main chain

Low-temperature polycondensation was used to obtain aromatic polyamides containing triarylmethane fragments in the main chain. It was shown that the synthesized aromatic polyamides are highly soluble in organic solvents. Transparent films were obtained from solutions of polyamides in dimethylformamide by casting. It was found that the resulting aromatic polyamides exhibit increased heat resistance, and films based on them have high mechanical properties.

Lowering trade barriers improves income distribution and economic resiliency

The U.S.-China trade war, COVID-19 pandemic and Russian invasion of Ukraine contributed to calls for greater economic self-sufficiency by exposing gaps in international collaboration and the uncertainty of global supply chains. In this study, we apply a comprehensive global modeling framework to enhance understanding of the potential impacts of fragmentation of global value chains. Supporting the general argument toward economic benefits from open markets our analysis contributes to the policy debate in two important dimensions of this phenomenon—distributional impacts and economic resiliency. We find that tariff liberalization and trade facilitation measures implemented by developing countries could not only reduce between-country inequality but also result in progressive within-country income distribution primarily through lowering food prices, as well as increasing unskilled wages. In addition, using a case study of disruption to Thailand’s electronics industry, we find higher resiliency of developing countries to external shocks in the globalized (as opposed to a localized) world.

Development of a Shigella conjugate vaccine targeting Shigella flexneri 6 that is immunogenic and provides protection against virulent challenge

Immunity protective against shigella infection targets the bacterial O-specific polysaccharide (OSP) component of lipopolysaccharide. A multivalent shigella vaccine would ideally target the most common global Shigella species and serotypes such as Shigella flexneri 2a, S. flexneri 3a, S. flexneri 6, and S. sonnei. We previously reported development of shigella conjugate vaccines (SCVs) targeting S. flexneri 2a (SCV-Sf2a) and 3a (SCV-Sf3a) using a platform squaric acid chemistry conjugation approach and carrier protein rTTHc, a 52 kDa recombinant protein fragment of the heavy chain of tetanus toxoid. Here we report development of a SCV targeting S. flexneri 6 (SCV-Sf6) using the same platform approach. We demonstrated that SCV-Sf6 was recognized by serotype-specific monoclonal antibodies and convalescent sera of humans recovering from shigellosis in Bangladesh, suggesting correct immunological display of OSP. We vaccinated mice and found induction of serotype-specific OSP and LPS IgG and IgM responses, as well as rTTHc-specific IgG responses. Immune responses were increased when administered with aluminum phosphate adjuvant. Vaccination induced bactericidal antibody responses against S. flexneri 6, and vaccinated animals were protected against lethal challenge with virulent S. flexneri 6. Our results assist in the development of a multivalent vaccine protective against shigellosis.

Chameleon Sequences-Structural Effects in Proteins Characterized by Hydrophobicity Disorder.

Repeated protein folding processes both in vivo and in vitro leading to the same structure for a specific amino acid sequence prove that the amino acid sequence determines protein structuring. This is also evidenced by the variability of structuring, dependent on the introduced mutations. An important phenomenon in this regard is the presence of a differentiated secondary structure for chain fragments of identical sequence representing distinct forms of the secondary-order structure. Proteins termed chameleon proteins contain polypeptide chain fragments of identical sequence (length 6-12 aa) showing structural differentiation: helix versus β-structure. In the present paper, it was shown that these fragments represent components matching the structural status dictated by the physicochemical properties of the entire structural unit. This structural matching is related to achieving the goal of the biological function of the structural unit. The corresponding secondary structure represents a means to achieving this goal, not an end in itself. A selected set of proteins from the ChSeq database have been analyzed using a fuzzy oil drop model (FOD-M) identifying the uniqueness of the hydrophobicity distribution taken as a medium for recording the specificity of a given protein and a given chameleon section in particular. It was shown that in the vast majority, the status of chameleon sections turns out to be comparable regardless of the represented secondary structure.

Motor innervation directs the correct development of the mouse sympathetic nervous system

The sympathetic nervous system controls bodily functions including vascular tone, cardiac rhythm, and the “fight-or-flight response”. Sympathetic chain ganglia develop in parallel with preganglionic motor nerves extending from the neural tube, raising the question of whether axon targeting contributes to sympathetic chain formation. Using nerve-selective genetic ablations and lineage tracing in mouse, we reveal that motor nerve-associated Schwann cell precursors (SCPs) contribute sympathetic neurons and satellite glia after the initial seeding of sympathetic ganglia by neural crest. Motor nerve ablation causes mispositioning of SCP-derived sympathoblasts as well as sympathetic chain hypoplasia and fragmentation. Sympathetic neurons in motor-ablated embryos project precociously and abnormally towards dorsal root ganglia, eventually resulting in fusion of sympathetic and sensory ganglia. Cell interaction analysis identifies semaphorins as potential motor nerve-derived signaling molecules regulating sympathoblast positioning and outgrowth. Overall, central innervation functions both as infrastructure and regulatory niche to ensure the integrity of peripheral ganglia morphogenesis.

Optimized inhaled LNP formulation for enhanced treatment of idiopathic pulmonary fibrosis via mRNA-mediated antibody therapy

Lipid nanoparticle-assisted mRNA inhalation therapy necessitates addressing challenges such as resistance to shear force damage, mucus penetration, cellular internalization, rapid lysosomal escape, and target protein expression. Here, we introduce the innovative “LOOP” platform with a four-step workflow to develop inhaled lipid nanoparticles specifically for pulmonary mRNA delivery. iLNP-HP08LOOP featuring a high helper lipid ratio, acidic dialysis buffer, and excipient-assisted nebulization buffer, demonstrates exceptional stability and enhanced mRNA expression in the lungs. By incorporating mRNA encoding IL-11 single chain fragment variable (scFv), scFv@iLNP-HP08LOOP effectively delivers and secretes IL-11 scFv to the lungs of male mice, significantly inhibiting fibrosis. This formulation surpasses both inhaled and intravenously injected IL-11 scFv in inhibiting fibroblast activation and extracellular matrix deposition. The HP08LOOP system is also compatible with commercially available ALC0315 LNPs. Thus, the “LOOP” method presents a powerful platform for developing inhaled mRNA nanotherapeutics with potential for treating various respiratory diseases, including idiopathic pulmonary fibrosis.

Carbon inequality under domestic value chain fragmentation

The division of production leads to the separation of production and consumption, resulting in carbon emissions transfer with trade among regions. Thus, quantifying carbon inequality caused by domestic trade is important for allocating emission-reduction tasks and improving emission-reduction efficiency. Considering domestic value chain (DVC) fragmentation in China, we use multiregional input–output tables to divide products into within-province trade products, traditional trade intermediate/final products, and DVC–related trade products. Then, we examine carbon intensity inequality (CII) under DVC fragmentation and, using Theil index to analyze the contributions of between-province economic differences, within-province trade structures, and related influencing factors to CII. Previous studies have mostly focused on issues related to global value chain fragmentation. We take the lead in exploring CII caused by DVC fragmentation, and for the first time decompose the composition of CII from the perspective of DVC fragmentation. In addition, the national CII is attempted to analyze its contribution at the provincial level for the first time, which is a new improvement and exploration of previous regional research perspectives. The empirical results show that China's CII is on an upward trend, with large changes between 2002 and 2007, and then stabilized between 2007 and 2010 due to the financial crisis, before entering a phase of rapid change after 2012. In terms of regional contribution, between-province inequality is the core to PCII, while within-province inequality is the key to CCII. In terms of province, the changes in between-province inequality are relatively stable at early period. In the middle and late stages, the temporal and spatial changes of PCII and CCII between provinces are obvious with “geographic stickiness” weakening. Specifically, the carbon transfer in the early study period is mostly concentrated in traditional trade products, and the share of carbon transfer in DVC-related trade products later rises significantly from 20.05% in 2002 to 36.32% in 2017, and the value-added transfer rises from 17.09% in 2002 to 27.65% in 2017. In terms of influencing factors, energy intensity remains an important component to CII. In addition, the uncertainties of the data and methodology used in this study are discussed and possible future research directions for expansion are elaborated. Finally, specific policy recommendations for mitigating carbon inequality are presented to inform the development of efficient carbon reduction strategies.

Dynamics of scFv-targeted VAP2 correlating with IL-16, MIF and IL-1Ra in ANCA-associated vasculitis

Background and hypothesisUsing a mouse model of MPA with microvascular lesion with a clone (VasSF) of recombinant single chain fragments of the variable region of human IgG, we previously showed that vasculitis-associated apolipoprotein A2 (VAP2) may be a therapeutic target for vasculitis. The present study estimated the target molecules for VasSF and the association between VAP2 and cytokine levels in patient sera in terms of microvascular lesion severity. MethodsSera and clinical information were collected from patients with microscopic polyangiitis and granulomatosis with polyangiitis (MPA/GPA) and infectious disease. Neutrophil counts, levels of C-reactive protein (CRP), creatinine, total cholesterol associated with microvascular lesion, HDL cholesterol, low-density lipoprotein cholesterol, triglycerides, glomerular filtration rate (eGFR), and cytokines were estimated. Serum VAP2 signals were determined with Western blotting. ResultsVasSF bound to a 24 kDa molecule in the serum of active MPA/GPA patients. Anti-AP2 antibody also bound with the same 24 kDa molecule, named VAP2, because of size difference from normal APOA2. The VAP2 signal was significantly stronger in the active-disease group but significantly weakened in remission. The signal correlated positively with eGFR but not with the Birmingham Vasculitis Activity Score, CRP, MPO-ANCA, or PR3-ANCA levels. It correlated negatively with MPO activity, IL-16, MIF, and IL-1Ra. Moreover, VasSF bound to a 17 kDa molecule in the remission phase. ConclusionThe 24 kDa VAP2 molecule may be associated with neutrophil functions because of its inverse correlation with MPO activity, IL-16, MIF, and IL-1Ra, suggesting that VAP2-APOA1 formation in HDL triggers microvascular injury. VasSF may reverse the injury by removing APOA1-VAP2 heterodimers from peripheral blood vessels.

Prevention of Blood Incompatibility Related Hemagglutination: Blocking of Antigen A on Red Blood Cells Using In Silico Designed Recombinant Anti-A scFv.

Critical blood shortages plague healthcare systems, particularly in lower-income and middle-income countries. This affects patients requiring regular transfusions and creates challenges during emergencies where universal blood is vital. To address these shortages and support blood banks during emergencies, this study reports a method for increasing the compatibility of blood group A red blood cells (RBCs) by blocking surface antigen-A using anti-A single chain fragment variable (scFv). To enhance stability, the scFv was first modified with the addition of interdomain disulfide bonds. The most effective location for this modification was found to be H44-L232 of mutant-1a scFv. ScFv was then produced from E.coli BL21(DE3) and purified using a three-step process. Purified scFvs were then used to block maximum number of antigens-A on RBCs, and it was found that only monomers were functional, while dimers formed through incorrect domain-swapping were non-functional. These antigen-blocked RBCs displayed no clumping in hemagglutination testing with incompatible blood plasma. The dissociation constant KD was found to be 0.724 μM. Antigen-blocked RBCs have the potential to be given to other blood groups during emergencies. This innovative approach could significantly increase the pool of usable blood, potentially saving countless lives.

Investigating the value of urinary biomarkers in relation to lupus nephritis histopathology: present insights and future prospects.

Lupus nephritis (LN), a leading cause of death in Systemic Lupus Erythematosus (SLE) patients, presents significant diagnostic and prognostic challenges. Although renal pathology offers critical insights regarding the diagnosis, classification, and therapy for LN, its clinical utility is constrained by the invasive nature and limited reproducibility of renal biopsies. Moreover, the continuous monitoring of renal pathological changes through repeated biopsies is impractical. Consequently, there is a growing interest in exploring urine as a non-invasive, easily accessible, and dynamic "liquid biopsy" alternative to guide clinical management. This paper examines novel urinary biomarkers from a renal pathology perspective, encompassing cellular components, cytokines, adhesion molecules, auto-antibodies, soluble leukocyte markers, light chain fragments, proteins, small-molecule peptides, metabolomics, urinary exosomes, and ribonucleic acids. We also discuss the application of combined models comprising multiple biomarkers in assessing lupus activity. These innovative biomarkers and models offer insights into LN disease activity, acute and chronic renal indices, fibrosis, thrombotic microangiopathy, podocyte injury, and other pathological changes, potentially improving the diagnosis, management, and prognosis of LN. These urinary biomarkers or combined models may serve as viable alternatives to traditional renal pathology, potentially revolutionizing the method for future LN diagnosis and observation.

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.