Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin

Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement

JABBERWOCK: A Tool for WebAssembly Dataset Generation and Its Application to Malicious Website Detection

Machine learning is often used for malicious website detection, but an approach incorporating WebAssembly as a feature has not been explored due to a limited number of samples, to the best of our knowledge. In this paper, we propose JABBERWOCK (JAvascript-Based Binary EncodeR by WebAssembly Optimization paCKer), a tool to generate WebAssembly datasets in a pseudo fashion via JavaScript. Loosely speaking, JABBERWOCK automatically gathers JavaScript code in the real world, convert them into WebAssembly, and then outputs vectors of the WebAssembly as samples for malicious website detection. We also conduct experimental evaluations of JABBERWOCK in terms of the processing time for dataset generation, comparison of the generated samples with actual WebAssembly samples gathered from the Internet, and an application for malicious website detection. Regarding the processing time, we show that JABBERWOCK can construct a dataset in 4.5 seconds per sample for any number of samples. Next, comparing 10,000 samples output by JABBERWOCK with 168 gathered WebAssembly samples, we believe that the generated samples by JABBERWOCK are similar to those in the real world. We then show that JABBERWOCK can provide malicious website detection with 99\% F1-score because JABBERWOCK makes a gap between benign and malicious samples as the reason for the above high score. We also confirm that JABBERWOCK can be combined with an existing malicious website detection tool to improve F1-scores. JABBERWOCK is publicly available via GitHub (https://github.com/c-chocolate/Jabberwock).Comment: Accepted in DCDS 2023 (co-located in DSN 2023

On the Black-Box Separation Between Ring Signatures and Public Key Encryptions

In this paper, we show that it is impossible to construct a public key encryption scheme (PKE) from a ring signature scheme in a black-box fashion in the standard model. Such an impossibility is highly non-trivial because, to the best of our knowledge, known generic constructions of ring signature scheme are based on public key cryptosystems or in the random oracle model. Technically, we introduce a new cryptographic primitive named indistinguishable multi-designated verifiers signature (IMDVS), and prove that (i) IMDVS is equivalent to PKE, and (ii) it is impossible to construct IMDVS from a ring signature scheme in a generic way. Our result suggests an essential gap between ring signature and group signature, as it is known that group signature implies PKE

On the Black-Box Impossibility of Multi-Designated Verifiers Signature Schemes from Ring Signature Schemes

From the work by Laguillaumie and Vergnaud in ICICS\u2704, it has been widely believed that multi-designated verifier signature schemes (MDVS) can be constructed from ring signature schemes in general. However in this paper, somewhat surprisingly, we prove that it is impossible to construct an MDVS scheme from a ring signature scheme in a black-box sense (in the standard model). The impossibility stems from the difference between the definitions of unforgeability. To the best of our knowledge, existing works demonstrating the constructions do not provide formal reduction from an MDVS scheme to a ring signature scheme, and thus the impossibility has been overlooked for a long time

A Coin-Free Oracle-Based Augmented Black Box Framework

After the work of Impagliazzo and Rudich (STOC, 1989), the black box framework has become one of the main research domain of cryptography. However black box techniques say nothing about non-black box techniques such as making use of zero-knowledge proofs. Brakerski et al. introduced a new black box framework named augmented black box framework, in which they gave a zero-knowledge proof oracle in addition to a base primitive oracle (TCC, 2011). They showed a construction of a non-interactive zero knowledge proof system based on a witness indistinguishable proof system oracle. They presented augmented black box construction of chosen ciphertext secure public key encryption scheme based on chosen plaintext secure public key encryption scheme and augmented black box separation between one-way function and key agreement. In this paper we simplify the work of Brakerski et al. by introducing a proof system oracle without witness indistinguishability, named coin-free proof system oracle, that aims to give the same construction and separation results of previous work. As a result, the augmented black box framework becomes easier to handle. Since our oracle is not witness indistinguishable, our result encompasses the result of previous work

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis

On the Black-Box impossibility of multi-designated verifiers signature schemes from ring signature schemes

From the work by Laguillaumie and Vergnaud in ICICS’04, it has been widely believed that multi-designated verifiers signature scheme (MDVS) can be constructed from ring signature schemes in general. However, in this article, somewhat surprisingly, we prove that it is impossible to construct an MDVS scheme from a ring signature scheme in a black-box sense (in the standard model). The impossibility stems from the difference between the definitions of unforgeability of the two schemes. To the best of our knowledge, existing works demonstrating the constructions do not provide formal reductions from an MDVS scheme to a ring signature scheme, and thus, the impossibility has been overlooked for a long time

STAP-2 positively regulates Fc epsilon RI-mediated basophil activation and basophil-dependent allergic inflammatory reactions

Basophils are an important cell type in the regulation of T(h)2 immune responses. Recently, we revealed that signal-transducing adaptor protein-2 (STAP-2) negatively regulates mast cell activation via Fc epsilon RI. However, the role of STAP-2 in basophil maturation and activation remained unclear. In this study, we demonstrated the normal development of basophils in STAP-2-deficient (STAP-2(-/-)) mice. We also demonstrated in vitro normal basophil differentiation and FceRI expression in STAP2(-/-) mice, suggesting that STAP-2 is dispensable for basophil maturation. Using bone marrow-derived cultured basophils (BMBs), we showed that degranulation and cytokine production of STAP-2(-/-) BMBs were lower than those of wild-type (WT) BMBs upon stimulation with IgE/Ag. In accordance with the reduction of degranulation and cytokine production, phosphorylation of several signal molecules such as Lyn, PLC-gamma 2 and Erk was reduced in STAP-2(-/-) BMBs after stimulation via Fc epsilon RI. Finally, it was observed that IgE-dependent chronic allergic inflammation of STAP-2(-/-) mice was significantly inhibited compared with WT mice. Taken together, we conclude that STAP-2 is an adaptor molecule that positively regulates FceRI-mediated basophil activation and basophil-dependent allergic inflammatory reactions

Iodine-123 β-methyl-P-iodophenyl-pentadecanoic Acid (¹²³I-BMIPP) Myocardial Scintigraphy for Breast Cancer Patients and Possible Early Signs of Cancer-Therapeutics-Related Cardiac Dysfunction (CTRCD)

(1) Background: The mortality of breast cancer has decreased due to the advancement of cancer therapies. However, more patients are suffering from cancer-therapeutics-related cardiac dysfunction (CTRCD). Diagnostic and treatment guidelines for CTRCD have not been fully established yet. Ultrasound cardiogram (UCG) is the gold standard for diagnosis of CTRCD, but many breast cancer patients cannot undergo UCG due to the surgery wounds or anatomical reasons. The purpose of the study is to evaluate the usefulness of myocardial scintigraphy using Iodine-123 β-methyl-P-iodophenyl-pentadecanoic acid (123I-BMIPP) in comparison with UCG. (2) Methods: 100 breast cancer patients who received chemotherapy within 3 years underwent Thallium (201Tl) and 23I-BMIPP myocardial perfusion and metabolism scintigraphy. The images were visually evaluated by doctors and radiological technologists, and the grade of uptake reduction was scored by Heart Risk View-S software (Nihon Medi-Physics). The scores were deployed in a 17-segment model of the heart. The distribution of the scores were analyzed. (3) Results: Nine patients (9%) could not undergo UCG. No correlation was found between left ventricular ejection fraction (LVEF) and Heart Risk View-S scores of 201Tl myocardial perfusion scintigraphy nor those of BMIPP myocardial metabolism scintigraphy. In a 17-segment model of the heart, the scores of the middle rings were higher than for the basal ring. (4) Conclusions: Evaluation by UCG is not possible for some patients. Myocardial scintigraphy cannot serve as a perfect alternative to UCG. However, it will become the preferable second-choice screening test, as it could point out the early stage of CTRCD