A novel bispecific antibody dual-targeting approach for enhanced neutralization against fast-evolving SARS-CoV-2 variants.

Introduction: The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has caused unprecedented health and socioeconomic crises, necessitating the immediate development of highly effective neutralizing antibodies. Despite recent advancements in anti-SARS-CoV-2 receptor-binding domain (RBD)-specific monoclonal antibodies (mAbs) derived from convalescent patient samples, their efficacy against emerging variants has been limited. In this study, we present a novel dual-targeting strategy using bispecific antibodies (bsAbs) that specifically recognize both the SARS-CoV-2 RBD and fusion peptide (FP), crucial domains for viral attachment to the host cell membrane and fusion in SARS-CoV-2 infection. Methods: Using phage display technology, we rapidly isolated FP-specific mAbs from an established human recombinant antibody library, identifying K107.1 with a nanomolar affinity for SARS-CoV-2 FP. Furthermore, we generated K203.A, a new bsAb built in immunoglobulin G4-(single-chain variable fragment)2 forms and demonstrating a high manufacturing yield and nanomolar affinity to both the RBD and FP, by fusing K102.1, our previously reported RBD-specific mAb, with K107.1. Results: Our comprehensive in vitro functional analyses revealed that the K203.A bsAb significantly outperformed the parental RBD-specific mAb in terms of neutralization efficacy against SARS-CoV-2 variants. Furthermore, intravenous monotherapy with K203.A demonstrated potent in vivo neutralizing activity without significant in vivo toxicity in a mouse model infected with a SARS-CoV-2 variant. Conclusion: These findings present a novel bsAb dual-targeting strategy, directed at SARS-CoV-2 RBD and FP, as an effective approach for rapid development and management against continuously evolving SARS-CoV-2 variants.

Estimation of Cooling Rate of High-Strength Thick Plate Steel during Water Quenching Based on a Dilatometric Experiment.

The microstructure and hardness along the thickness direction of a water-quenched, high-strength thick plate with a thickness of 40 mm were investigated with three specimens from the thick plate: surface, 1/4t, and 1/2t (center) thickness, and the phase transformation behavior of the thick plate according to the cooling rate was analyzed through dilatometric experiments. Finally, the cooling rate for each thickness of the thick plate was estimated by comparing the microstructure and hardness of the thick plate along with the thickness with those of the dilatometric specimens. Martensite microstructure was observed on the surface of the water-quenched thick plate due to the fast cooling rate. On the other hand, an inhomogeneous microstructure was transformed inside the thick plate due to the relatively slow cooling rate and central segregation of Mn. A small fraction of bainite was shown at 1/4t thickness. A banded microstructure with martensite and bainite resulting from Mn segregation was developed at 1/2t; that is, the full martensite microstructure was transformed in the Mn-enriched area even at a slow cooling rate due to high hardenability, but a bainite microstructure was formed in the Mn-depleted area owing to relatively low hardenability. A portion of martensite with fine cementite at the surface and 1/4t was identified as auto-tempered martensite with a Bagaryatskii orientation relationship between the ferrite matrix and cementite. The microstructure and hardness as well as dilatation were investigated at various cooling rates through a dilatometric experiment, and a continuous cooling transformation (CCT) diagram was finally presented for the thick plate. Comparing the microstructure and hardness at the surface, 1/4t, and 1/2t of the thick plate with those of dilatometric specimens cooled at various cooling rates, it was estimated that the surface of the thick plate was cooled at more than 20 °C/s, whereas the 1/4t region was cooled at approximately 5~10 °C/s during water quenching. Despite the difficulty in estimation of the cooling rate of 1/2t due to the banded structure, the cooling rate of 1/2t was estimated between 3 and 5 °C/s based on the results of an Mn-depleted zone.

Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody.

Signal transducer and activator of transcription 3 (STAT3) is a multifunctional protein that participates in signaling pathways initiated by various growth factors and cytokines. It exists in multiple forms including those phosphorylated on Tyr(705) (pYSTAT3) or Ser(727) (pSSTAT3) as well as the unphosphorylated protein (USTAT3). In addition to the canonical transcriptional regulatory role of pYSTAT3, both USTAT3 and pSSTAT3 function as transcriptional regulators by binding to distinct promoter sites and play signaling roles in the cytosol or mitochondria. The roles of each STAT3 species in different biological processes have not been readily amenable to investigation, however. We have now prepared an intrabody that binds specifically and with high affinity to the tyrosine-phosphorylated site of pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells as well as mouse liver blocked both the accumulation of pYSTAT3 in the nucleus and the production of acute phase response proteins induced by interleukin-6. Intrabody expression did not affect the overall accumulation of pSSTAT3 induced by interleukin-6 or phorbol 12-myristate 13-acetate (PMA), the PMA-induced expression of the c-Fos gene, or the PMA-induced accumulation of pSSTAT3 specifically in mitochondria. In addition, it had no effect on interleukin-6-induced expression of the gene for IFN regulatory factor 1, a downstream target of STAT1. Our results suggest that the engineered intrabody is able to block specifically the downstream effects of pYSTAT3 without influencing those of pSSTAT3, demonstrating the potential of intrabodies as tools to dissect the cellular functions of specific modified forms of proteins that exist as multiple species.

The effects of pressure on arthritic knees in a rat model of CFA-induced arthritis.

BACKGROUND: Pain is influenced by weather changes under certain circumstances, and inflammatory pain in animal models is ameliorated by pressure, but the underlying mechanism of atmospheric pressure has not been clearly elucidated. OBJECTIVE: To examine the effect of pressure on pain in an arthritic animal model. STUDY DESIGN: Controlled animal study. SETTING: Laboratory animal study. METHODS: Following an injection of complete Freund's adjuvant (CFA) into one side of a knee joint, 32 rats were assigned randomly to 2 groups and either placed under 1 or 2.5 atmospheres absolute (ATA) in a hyperbaric chamber for 5 hours. The pain levels were assessed daily for up to 2 weeks post-injection to determine the changes in weight bearing (WB) of the affected limbs. In addition, the levels of gelatinase, MMP-2, and MMP-9 expression in the synovial fluids of the knees were analyzed. RESULTS: After arthritis induction, the rats in the 1 ATA group showed reduced WB of the affected limbs (< 10% of normal limbs). This reduction in WB peaked at 2 days after the injection and then decreased spontaneously. Nevertheless, the pain behavior lasted for more than 2 weeks. In the 2.5 ATA group, the WB was significantly better during the experiment.  The MMP-9/MMP-2 ratio increased at 7 and 14 days after the CFA injection in the 1 ATA group. However, repetitive exposure to 2.5 ATA significantly reduced this ratio in the 2.5 ATA group. LIMITATIONS: Although a sufficient number of samples were used to support the hypothesis that high atmospheric pressure improves a painful condition in this study, an additional larger-scale study will be needed to confirm these findings. CONCLUSION: Exposure to elevated pressures appears to relieve arthritic pain for extended periods by reducing the inflammatory process and should be considered as a possible alternative pain-reducing therapy.

5-(4-Hydroxy-2,3,5-trimethylbenzylidene) thiazolidine-2,4-dione attenuates atherosclerosis possibly by reducing monocyte recruitment to the lesion.

A variety of benzylidenethiazole analogs have been demonstrated to inhibit 5-lipoxygenase (5-LOX). Here we report the anti-atherogenic potential of 5-(4-hydroxy- 2,3,5-trimethylbenzylidene) thiazolidin-2,4-dione (HMB-TZD), a benzylidenethiazole analog, and its potential mechanism of action in LDL receptor-deficient (Ldlr-/-) mice. HMB-TZD Treatment reduced leukotriene B4 (LTB4) production significantly in RAW264.7 macrophages and SVEC4-10 endothelial cells. Macrophages or endothelial cells pre-incubated with HMB-TZD for 2 h and then stimulated with lipopolysaccharide or tumor necrosis factor-alpha (TNF-α) displayed reduced cytokine production. Also, HMB-TZD reduced cell migration and adhesion in accordance with decreased proinflammatory molecule production in vitro and ex vivo. HMB-TZD treatment of 8-week-old male Ldlr-/- mice resulted in significantly reduced atherosclerotic lesions without a change to plasma lipid profiles. Moreover, aortic expression of pro-atherogenic molecules involved in the recruitment of monocytes to the aortic wall, including TNF-α , MCP-1, and VCAM-1, was downregulated. HMB-TZD also reduced macrophage infiltration into atherosclerotic lesions. In conclusion, HMB-TZD ameliorates atherosclerotic lesion formation possibly by reducing the expression of proinflammatory molecules and monocyte/macrophage recruitment to the lesion. These results suggest that HMB-TZD, and benzylidenethiazole analogs in general, may have therapeutic potential as treatments for atherosclerosis.

Platelet serotonin transporter function after short-term paroxetine treatment in patients with panic disorder.

Dysfunctions in serotonin neurotransmission have been implicated in some psychiatric disorders, and in particular, altered serotonin transporter function has been noted in panic disorder. In this study, the authors compared platelet [(3)H]serotonin uptake parameters, including maximum velocities (V(max)) and affinity constants (K(m)), in patients with panic disorder not undergoing treatment (n=21) and in healthy subjects (n=20). V(max) and K(m) values were re-examined after 12 weeks of paroxetine treatment. Values of V(max) and K(m) were lower in panic disorder patients at baseline than in healthy subjects. After treatment, K(m) normalized in panic patients, whereas V(max) did not change. A significant inverse correlation was found between increased K(m) and changes in anxiety levels. These results support a hypothesis of serotonergic transporter abnormalities in panic disorder, and suggest that increased K(m) values of platelet serotonin transporters parallel clinical improvement after short-term pharmacotherapy in panic disorder.

Sympathetic nervous function and the effect of the catechol-O-methyltransferase Val(158)Met polymorphism in patients with panic disorder.

BACKGROUND: Sympathetic nervous function abnormalities have long been suggested to be a possible etiology of panic disorder (PD). Catechol-O-methyltransferase (COMT) affects sympathetic activities, and the COMT Val(158)Met polymorphism has been suggested to be related to PD. The authors examined the relationship between sympathetic nervous function and the COMT Val(158)Met polymorphism in PD patients. METHODS: Fifty-eight patients [Val/Val (51.7%) and Met allele carriers (48.3%)] and 58 age-matched normal control subjects [Val/Val (56.9%) and Met allele carriers (43.1%)] were compared in terms of finger skin temperature, which is known to be a useful marker of sympathetic nervous function. RESULTS: A significant COMT Val(158)Met polymorphismxdiagnosis interaction was found. Specifically, the met allele was found to be associated with a lower skin temperature in PD patients. CONCLUSION: These results suggest that the COMT Met allele is related to the higher sympathetic nervous function observed in PD.