Sphingolipid long-chain base hydroxylation influences plant growth and callose deposition in Physcomitrium patens.

Sphingolipids are enriched in microdomains in the plant plasma membrane (PM). Hydroxyl groups in the characteristic long-chain base (LCB) moiety might be essential for the interaction between sphingolipids and sterols during microdomain formation. Investigating LCB hydroxylase mutants in Physcomitrium patens might therefore reveal the role of certain plant sphingolipids in the formation of PM subdomains. Physcomitrium patens mutants for the LCB C-4 hydroxylase S4H were generated by homologous recombination. Plants were characterised by analysing their sphingolipid and steryl glycoside (SG) profiles and by investigating different gametophyte stages. s4h mutants lost the hydroxyl group at the C-4 position of their LCB moiety. Loss of this hydroxyl group caused global changes in the moss sphingolipidome and in SG composition. Changes in membrane lipid composition may trigger growth defects by interfering with the localisation of membrane-associated proteins that are crucial for growth processes such as signalling receptors or callose-modifying enzymes. Loss of LCB-C4 hydroxylation substantially changes the P. patens sphingolipidome and reveals a key role for S4H during development of nonvascular plants. Physcomitrium patens is a valuable model for studying the diversification of plant sphingolipids. The simple anatomy of P. patens facilitates visualisation of physiological processes in biological membranes.

Effect of Erythropoietin on Morphofunctional Properties of Mesenchymal Stem Cells.

We studied the effect of erythropoietin on the morphofunctional status of bone marrow mesenchymal stem cells in patients with coronary heart disease. It was shown that the duration of cell exposure with erythropoietin had different effects on the expression levels of adhesion molecules, erythropoietin receptors, and co-expression of the erythropoietin receptor and common ß-chain of cytokines, apoptosis/necrosis, and the cell cycle. In most cases, erythropoietin increased proliferation, migration, and NO production by "aged" mesenchymal stem cells (passage 8) and passage 4 mesenchymal stem cells grown during the previous 3 passages in the presence of 33.4 U/ml erythropoietin. Erythropoietin increased the expression of the autophagy marker LC3B in mesenchymal stem cells grown in the presence of erythropoietin in the culture medium. Thus, long-term culturing of mesenchymal stem cells in the presence of erythropoietin in the culture medium increased their resistance to adverse microenvironment factors - oxidative stress and hyperglycemia.

Lignocellulosic biomass: Hurdles and challenges in its valorization.

Lignocellulosic biomass (LCB) is globally available and sustainable feedstock containing sugar-rich platform that can be converted to biofuels and specialty products through appropriate processing. This review focuses on the efforts required for the development of sustainable and economically viable lignocellulosic biorefinery to produce carbon neutral biofuels along with the specialty chemicals. Sustainable biomass processing is a global challenge that requires the fulfillment of fundamental demands concerning economic efficiency, environmental compatibility, and social responsibility. The key technical challenges in continuous biomass supply and the biological routes for its saccharification with high yields of sugar sources have not been addressed in research programs dealing with biomass processing. Though many R&D endeavors have directed towards biomass valorization over several decades, the integrated production of biofuels and chemicals still needs optimization from both technical and economical perspectives. None of the current pretreatment methods has advantages over others since their outcomes depend on the type of feedstock, downstream process configuration, and many other factors. Consolidated bio-processing (CBP) involves the use of single or consortium of microbes to deconstruct biomass without pretreatment. The use of new genetic engineering tools for natively cellulolytic microbes would make the CBP process low cost and ecologically friendly. Issues arising with chemical characteristics and rigidity of the biomass structure can be a setback for its viability for biofuel conversion. Integration of functional genomics and system biology with synthetic biology and metabolic engineering undoubtedly led to generation of efficient microbial systems, albeit with limited commercial potential. These efficient microbial systems with new metabolic routes can be exploited for production of commodity chemicals from all the three components of biomass. This paper provides an overview of the challenges that are faced by the processes converting LCB to commodity chemicals with special reference to biofuels.

Pharmacokinetics, Pharmacodynamics, and Tolerability of Single-Dose Oral LCB01-0371, a Novel Oxazolidinone with Broad-Spectrum Activity, in Healthy Volunteers.

LCB01-0371 is a novel oxazolidinone with broad-spectrum activity against Gram-positive pathogens in both in vitro studies and animal infection models. The objectives of this study were to evaluate its safety, tolerability, pharmacokinetics, and pharmacodynamics following single ascending doses. Single oral doses of 600 mg linezolid, a placebo, or LCB01-0371 of between 50 mg and 3,200 mg were tested in 69 healthy male subjects. Blood and urine were sampled, LCB01-0371 concentrations were measured, and the serum inhibitory and bactericidal titers of LCB01-0371 and linezolid were determined. LCB01-0371 was well tolerated up to 2,400 mg. The most common drug-related clinical and laboratory adverse events were nausea with or without vomiting, decreased neutrophil counts, and increased total bilirubin levels. The frequency of adverse events and drug-related adverse events was similar among the treatment groups. The systemic exposure was approximately dose proportional over the range of 50 mg to 800 mg, which includes the anticipated clinical dose. The mean clearance, renal clearance, and volume of distribution were significantly decreased at higher doses (above 800 mg). LCB01-0371 exhibited early bacteriostatic activity against all tested strains except for Streptococcus pneumoniae strains, and the potency of LCB01-0371 at 800 mg was similar to that of linezolid at the therapeutic dose (600 mg). However, LCB01-0371 had less bactericidal activity than linezolid. Taken together, LCB01-0371 was well tolerated, exhibited approximate dose proportionality within the anticipated clinically relevant dose range, and showed bacteriostatic and bactericidal activity comparable to that of linezolid. These results support the further clinical development of LCB01-0371. (This study has been registered at ClinicalTrials.gov under registration no. NCT01554995.).

Activity of LCB01-0371, a Novel Oxazolidinone, against Mycobacterium abscessus.

Mycobacterium abscessus is a highly pathogenic drug-resistant rapidly growing mycobacterium. In this study, we evaluated the in vitro, intracellular, and in vivo activities of LCB01-0371, a novel and safe oxazolidinone derivative, for the treatment of M. abscessus infection and compared its resistance to that of other oxazolidinone drugs. LCB01-0371 was effective against several M. abscessus strains in vitro and in a macrophage model of infection. In the murine model, a similar efficacy to linezolid was achieved, especially in the lungs. We induced laboratory-generated resistance to LCB01-0371; sequencing analysis revealed mutations in rplC of T424C and G419A and a nucleotide insertion at the 503 position. Furthermore, LCB01-0371 inhibited the growth of amikacin-, cefoxitin-, and clarithromycin-resistant strains. Collectively, our data indicate that LCB01-0371 might represent a promising new class of oxazolidinones with improved safety, which may replace linezolid for the treatment of M. abscessus.

In Vivo Activity of LCB 01-0699, a Prodrug of LCB 01-0648, against Staphylococcus aureus.

LCB01-0648 is a novel oxazolidinone compound that shows potent antibacterial activities against most Gram-positive cocci, including the multi-drug resistant Staphylococcusaureus. In this study, in vivo activity of LCB01-0699, a LCB01-0648 prodrug, against S.aureus was evaluated in comparison with that of Linezolid. The results of the systemic infection study demonstrated that LCB01-0699 was more potent than Linezolid against methicillin-susceptible and -resistant S. aureus strains. The in vivo efficacy of LCB01-0699 against methicillin-susceptible and -resistant S. aureus strains in a skin infection model showed more potent activity than Linezolid. LCB01-0699 shows potent in vivo activity against methicillin-susceptible and -resistant S. aureus strains, suggesting that LCB01-0699 would be a novel candidate for the treatment of these infectious diseases caused by S. aureus.

In Vitro Activities of LCB 01-0648, a Novel Oxazolidinone, against Gram-Positive Bacteria.

Oxazolidinones are a novel class of synthetic antibacterial agents that inhibit bacterial protein synthesis. Here, we synthesized and tested a series of oxazolidinone compounds containing cyclic amidrazone. Among these compounds, we further investigated the antibacterial activities of LCB01-0648 against drug-susceptible or resistant Gram-positive cocci in comparison with those of six reference compounds. LCB01-0648 showed the most potent antimicrobial activities against clinically isolated Gram-positive bacteria. Against the methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCNS) isolates, LCB01-0648 showed the lowest MIC90s (0.5 mg/L) among the tested compounds. In addition, LCB01-0648 had the lowest minimum inhibitory concentrations (MICs) against the four linezolid-resistant S. aureus (LRSA) strains (range 2-4 mg/L). The results of the time-kill studies demonstrated that LCB01-0648 at a concentration 8× the (MIC) showed bactericidal activity against methicillin-susceptible Staphylococcus aureus MSSA or MRSA, but showed a bacteriostatic effect against LRSA. These results indicate that LCB01-0648 could be a good antibacterial candidate against multidrug-resistant (MDR) Gram-positive cocci.

Stomatal closure induced by phytosphingosine-1-phosphate and sphingosine-1-phosphate depends on nitric oxide and pH of guard cells in Pisum sativum.

MAIN CONCLUSION: Phyto-S1P and S1P induced stomatal closure in epidermis of pea ( Pisum sativum ) by raising the levels of NO and pH in guard cells. Phosphosphingolipids, such as phytosphingosine-1-phosphate (phyto-S1P) and sphingosine-1-phosphate (S1P), are important signaling components during drought stress. The biosynthesis of phyto-S1P or S1P is mediated by sphingosine kinases (SPHKs). Although phyto-S1P and S1P are known to be signaling components in higher plants, their ability to induce stomatal closure has been ambiguous. We evaluated in detail the effects of phyto-S1P, S1P and SPHK inhibitors on signaling events leading to stomatal closure in the epidermis of Pisum sativum. Phyto-S1P or S1P induced stomatal closure, along with a marked rise in nitric oxide (NO) and cytoplasmic pH of guard cells, as in case of ABA. Two SPHK inhibitors, DL-threo dihydrosphingosine and N',N'-dimethylsphingosine, restricted ABA-induced stomatal closure and prevented the increase of NO or pH by ABA. Modulators of NO or pH impaired both stomatal closure and increase in NO or pH by phyto-S1P/S1P. The stomatal closure by phyto-S1P/S1P was mediated by phospholipase D and phosphatidic acid (PA). When present, PA elevated the levels of pH, but not NO of guard cells. Our results demonstrate that stomatal closure induced by phyto-S1P and S1P depends on rise in pH as well as NO of guard cells. A scheme of signaling events initiated by phyto-S1P/S1P, and converging to cause stomatal closure, is proposed.

Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells.

The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.

Anti-angiogenic activity of thienopyridine derivative LCB03-0110 by targeting VEGFR-2 and JAK/STAT3 Signalling.

Vascular endothelial growth factor receptor-2 (VEGFR-2) and Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signalling are important for tumor angiogenesis and metastasis. In this study, we identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB03-0110) as a potent angiogenesis inhibitor. LCB03-0110 inhibited VEGFR-2 and JAK/STAT3 signalling in primary cultured human endothelial cells and cancer cells. An in vitro kinase assay and molecular modelling revealed that LCB03-0110 inhibited VEGFR-2, c-SRC and TIE-2 kinase activity via preferential binding at the ATP-binding site of their kinases. LCB03-0110 successfully occupied the hydrophobic pocket of VEGFR-2, c-SRC and TIE-2. LCB03-0110 also inhibited hypoxia-induced HIF/STAT3 and EGF- or angiopoietin-induced signalling cascades. In addition, LCB03-0110 inhibited VEGF-induced proliferation, viability, migration and capillary-like tube formation. LCB03-0110 also suppressed the sprouting of endothelial cells in the rat aorta and the formation of new blood vessels in the mouse Matrigel plug assay, but also suppressed pulmonary metastasis and tumor xenograft in mice. Our results suggest that LCB03-0110 is a potential candidate small molecule for blocking angiogenesis mediated by aberrant activation of VEGFR-2 and JAK/STAT3 signalling.

Protective Effects of Isoliquiritigenin and Licochalcone B on the Immunotoxicity of BDE-47: Antioxidant Effects Based on the Activation of the Nrf2 Pathway and Inhibition of the NF-κB Pathway.

2,2',4,4'-Tetrabrominated biphenyl ether (BDE-47) is a polybrominated diphenyl ether (PBDE) homologue that is ubiquitous in biological samples and highly toxic to humans and other organisms. Prior research has confirmed that BDE-47 can induce oxidative damage in RAW264.7 cells, resulting in apoptosis and impaired immune function. The current study mainly focused on how Isoliquiritigenin (ISL) and Licochalcone B (LCB) might protect against BDE-47's immunotoxic effects on RAW264.7 cells. The results show that ISL and LCB could increase phagocytosis, increase the production of MHC-II, and decrease the production of inflammatory factors (TNF-α, IL-6, and IL-1ß) and co-stimulatory factors (CD40, CD80, and CD86), alleviating the immune function impairment caused by BDE-47. Secondly, both ISL and LCB could reduce the expressions of the proteins Bax and Caspase-3, promote the expression of the protein Bcl-2, and reduce the apoptotic rate, alleviating the apoptosis initiated by BDE-47. Additionally, ISL and LCB could increase the levels of antioxidant substances (SOD, CAT, and GSH) and decrease the production of reactive oxygen species (ROS), thereby counteracting the oxidative stress induced by BDE-47. Ultimately, ISL and LCB suppress the NF-κB pathway by down-regulating IKBKB and up-regulating IκB-Alpha in addition to activating the Nrf2 pathway and promoting the production of HO-1 and NQO1. To summarize, BDE-47 causes oxidative damage that can be mitigated by ISL and LCB through the activation of the Nrf2 pathway and inhibition of the NF-κB pathway, which in turn prevents immune function impairment and apoptosis. These findings enrich the current understanding of the toxicological molecular mechanism of BDE-47 and the detoxification mechanism of licorice.

A comprehensive review on anaerobic digestion with focus on potential feedstocks, limitations associated and recent advances for biogas production.

With the escalating energy demand to accommodate the growing population and its needs along with the responsibility to mitigate climate change and its consequences, anaerobic digestion (AD) has become the potential approach to sustainably fulfil our demands and tackle environmental issues. Notably, a lot of attention has been drawn in recent years towards the production of biogas around the world in waste-to-energy perspective. Nevertheless, the progress of AD is hindered by several factors such as operating parameters, designing and the performance of AD reactors. Furthermore, the full potential of this approach is not fully realised yet due the dependence on people's acceptance and government policies. This article focuses on the different types of feedstocks and their biogas production potential. The feedstock selection is the basic and most important step for accessing the biogas yield. Furthermore, different stages of the AD process, design and the configuration of the biogas digester/reactors have been discussed to get better insight into process. The important aspect to talk about this process is its limitations associated which have been focused upon in detail. Biogas is considered to attain the sustainable development goals (SDG) proposed by United Nations. Therefore, the huge focus should be drawn towards its improvements to counter the limitation and makes it available to all the rural communities in developing countries and set-up the pilot scale AD plants in both developing and developed countries. In this regard, this article talks about the improvements and futures perspective related to the AD process and biogas enhancement.

ER Membrane Lipid Composition and Metabolism: Lipidomic Analysis.

Plant ER membranes are the major site of biosynthesis of several lipid families (phospholipids, sphingolipids, neutral lipids such as sterols and triacylglycerols). The structural diversity of lipids presents considerable challenges to comprehensive lipid analysis. This chapter will briefly review the various biosynthetic pathways and will detail several aspects of the lipid analysis: lipid extraction, handling, separation, detection, identification, and data presentation. The different tools/approaches used for lipid analysis will also be discussed in relation to the studies to be carried out on lipid metabolism and function.

Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway.

Sphingosine 1-phosphate (S1P) plays important roles both as a bioactive lipid molecule and an intermediate of the sphingolipid-to-glycerophospholipid metabolic pathway. To identify human acyl-CoA synthetases (ACSs) involved in S1P metabolism, we cloned all 26 human ACS genes and examined their abilities to restore deficient sphingolipid-to-glycerophospholipid metabolism in a yeast mutant lacking two ACS genes, FAA1 and FAA4. Here, in addition to the previously identified ACSL family members (ACSL1, 3, 4, 5, and 6), we found that ACSVL1, ACSVL4, and ACSBG1 also restored metabolism. All 8 ACSs were localized either exclusively or partly to the endoplasmic reticulum (ER), where S1P metabolism takes place. We previously proposed the entire S1P metabolic pathway from results obtained using yeast cells, i.e., S1P is metabolized to glycerophospholipids via trans-2-hexadecenal, trans-2-hexadecenoic acid, trans-2-hexadecenoyl-CoA, and palmitoyl-CoA. However, as S1P is not a naturally occurring long-chain base 1-phosphate in yeast, the validity of this pathway required further verification using mammalian cells. In the present study, we treated HeLa cells with the ACS inhibitor triacsin C and found that inhibition of ACSs resulted in accumulation of trans-2-hexadecenoic acid as in ACS mutant yeast. From these results, we conclude that S1P is metabolized by a common pathway in eukaryotes.

Development of highly effective LCB1-based lipopeptides targeting the spike receptor-binding motif of SARS-CoV-2.

LCB1 is a computationally designed 56-mer miniprotein targeting the spike (S) receptor-binding motif of SARS-CoV- 2 with high potent activity (Science, 2020; Cell host microbe, 2021); however, recent studies have demonstrated that emerging SARS-CoV-2 variants are highly resistant to LCB1's inhibition. In this study, we first identified a truncated peptide termed LCB1v8, which maintained the high antiviral potency. Then, a group of lipopeptides were generated by modifying LCB1v8 with diverse lipids, and of two lipopeptides, the C-terminally stearicacid-conjugtaed LCB1v17 and cholesterol-conjugated LCB1v18, were highly effective in inhibiting both S protein-pseudovirus and authentic SARS-CoV-2 infections. We further showed that LCB1-based inhibitors had similar α-helicity and thermostability in structure and bound to the target-mimic RBD protein with high affinity, and the lipopeptides exhibited greatly enhanced binding with the viral and cellular membranes, improved inhibitory activities against emerging SARS-CoV-2 variants. Moreover, LCB1v18 was validated with high preventive and therapeutic efficacies in K18-hACE2 transgenic mice against lethal SARS-CoV-2 challenge. In conclusion, our studies have provided important information for understanding the structure and activity relationship (SAR) of LCB1 inhibitor and would guide the future development of novel antivirals.

Oral Administration of Lacticaseibacillus rhamnosus CRL1505 Modulates Lung Innate Immune Response against Klebsiella pneumoniae ST25.

Orally administered Lacticaseibacillus rhamnosus CRL1505 enhances respiratory immunity, providing protection against respiratory viruses and Streptococcus pneumoniae. However, the capacity of the CRL1505 strain to improve respiratory immunity against Gram-negative bacterial infections has not been evaluated before. The aim of this work was to evaluate whether the Lcb. rhamnosus CRL1505 was able to beneficially regulate the respiratory innate immune response and enhance the resistance to hypermucoviscous KPC-2-producing Klebsiella pneumoniae of the sequence type 25 (ST25). BALB/c mice were treated with the CRL1505 strain via the oral route and then nasally challenged with K. pneumoniae ST25 strains LABACER 01 or LABACER 27. Bacterial cell counts, lung injuries and the respiratory and systemic innate immune responses were evaluated after the bacterial infection. The results showed that K. pneumoniae ST25 strains increased the levels of TNF-α, IL-1ß, IL-6, IFN-γ, IL-17, KC and MPC-1 in the respiratory tract and blood, as well as the numbers of BAL neutrophils and macrophages. Mice treated with Lcb. rhamnosus CRL1505 had significantly lower K. pneumoniae counts in their lungs, as well as reduced levels of inflammatory cells, cytokines and chemokines in the respiratory tract and blood when compared to infected controls. Furthermore, higher levels of the regulatory cytokines IL-10 and IL-27 were found in the respiratory tract and blood of CRL1505-treated mice than controls. These results suggest that the ability of Lcb. rhamnosus CRL1505 to help with the control of detrimental inflammation in lungs during K. pneumoniae infection would be a key feature to improve the resistance to this pathogen. Although further mechanistic studies are necessary, Lcb. rhamnosus CRL1505 can be proposed as a candidate to improve patients' protection against hypermucoviscous KPC-2-producing strains belonging to the ST25, which is endemic in the hospitals of our region.

Ruminal content biochar supplementation for enhanced biomethanation of rice straw: Focusing on biochar characterization and dose optimization.

Anaerobic digestion (AD) of agricultural wastes is a promising approach for energy recovery and crop residue management. However, its recalcitrant chemical structure hinders microbial hydrolysis and reduces biomethane production under AD. Biochar supplementation has been proven to promote the digestibility and biomethanation of lignocellulosic substrates. Therefore, this study investigated the influence of different pyrolysis temperatures (450 °C, 550 °C, and 650 °C) on the physicochemical properties of biochar. Furthermore, the impact of ruminal content biochar supplementation (1 %, 2 %, and 3 %) on the AD of rice straw with rumen fluid as inoculum has been investigated. The ruminal content biochar (RUCB) supplemented reactors showed an increment in biomethane yield and the highest cumulative biomethane yield 243.11 mL/g volatile solids (VS)) was recorded at 2 % RUCB supplementation, followed by 227.12 mL/g VS at 1 % RUCB supplementation and 162.86 mL/g VS at 3 % RUCB supplementation (P > 0.05). Compared to the control reactors (128.68 mL/g VS), RUCB supplemented reactors exhibited 1.88-fold, 1.76-fold, and 1.26-fold increments in biomethane yield due to pH stabilization and facilitation of microbial biofilm formation on the biochar. The correlation analysis showed that biomethane production is positively correlated with VS reduction (R2 = 0.9852). This study proposed a potential strategy to utilize ruminal content waste as a feedstock for biochar production and its application in AD for accelerating the biomethanation of rice straw.

On the resistance of new lightweight block ciphers against differential cryptanalysis.

Many recently proposed lightweight block ciphers lack security evaluation against generic cryptanalytic attacks such as differential cryptanalysis. In this paper, we contribute towards security evaluation efforts by investigating four lightweight Feistel-based block ciphers including SLIM, LBC-IoT, SCENERY, and LCB. SLIM claims resistance to differential cryptanalysis since, using a heuristic technique, its designers could only find a 7-round differential trail. Despite having no analysis of security against attacks such as differential cryptanalysis, the designers of LBC-IoT and LCB claimed that their ciphers are secure. Meanwhile, the designers of SCENERY claim that the best 11-round differential trail for the cipher has a probability of 2 - 66 . To substantiate these claims, we propose attacks on all four ciphers based on differential cryptanalysis. We presented practical key recovery attacks on SLIM which can retrieve the final round key for up to 14 rounds with a time complexity of 232. LBC-IoT was found to be weaker against differential cryptanalysis despite sharing many similarities with SLIM, whereby a key recovery attack of up to 19 rounds is possible with time complexity 231. For SCENERY, we found a differential trail of up to 12 rounds with probability 2 - 60 , which was used as the distinguisher for a 13-round key recovery attack. We also discovered that LCB's design lacks nonlinearity, allowing us to easily derive deterministic differential trails regardless of the number of rounds. This flaw allowed us to perform a trivial distinguishing attack using a single known ciphertext. By using a different S-box to address this flaw, LCB is now more resilient to differential cryptanalysis than SLIM and LBC-IoT when using the same number of rounds. Our paper presents new independent cryptanalysis results for these ciphers.

Susceptibility and Resistance of SARS-CoV-2 Variants to LCB1 and Its Multivalent Derivatives.

LCB1 is a computationally designed three-helix miniprotein that precisely targets the spike (S) receptor-binding motif (RBM) of SARS-CoV-2, exhibiting remarkable antiviral efficacy; however, emerging SARS-CoV-2 variants could substantially compromise its neutralization effectiveness. In this study, we constructed two multivalent LCB1 fusion proteins termed LCB1T and LCB1T-Fc, and characterized their potency in inhibiting SARS-CoV-2 pseudovirus and authentic virus in vitro. In the inhibition of various SARS-CoV-2 variants, the two LCB1 fusion proteins exhibited markedly improved inhibitory activities compared to LCB1 as anticipated; however, it was observed that relative to the D614G mutation hosting variant, the variants Delta, Lambda, and Omicron BQ.1.1, XBB, XBB.1.5, and EG.5.1 caused various degrees of resistance to the two fusion proteins' inhibition, with XBB, XBB.1.5, and EG.5.1 variants showing high-level resistance. Moreover, we demonstrated that bat coronavirus RaTG13 and pangolin coronavirus PCoV-GD/PCoV-GX were highly sensitive to two LCB1 fusion proteins, but not LCB1, inhibition. Importantly, our findings revealed a notable decrease in the blocking capacity of the multivalent LCB1 inhibitor on the interaction between the virus's RBD/S and the cell receptor ACE2 when confronted with the XBB variant compared to WT and the Omicron BA.1 variant. In conclusion, our studies provide valuable insights into the antiviral profiling of multivalent LCB1 inhibitors and offer a promising avenue for the development of novel broad-spectrum antiviral therapeutics.

Total Chemical Synthesis of a SARS-CoV-2 Miniprotein Inhibitor LCB1.

Native chemical ligation is a widely used technique for peptide fragment condensation in aqueous solutions, which has broken through the length limitation of traditional solid-phase peptide synthesis. It can achieve high-efficient chemical synthesis of proteins containing more than 300 amino acid residues. Peptide hydrazide, as a valuable reagent equivalent to a thioester peptide, can be easily and efficiently prepared by the Fmoc-based SPPS method and has been widely used in native chemical ligation. Here we take the chemical synthesis of a SARS-CoV-2 miniprotein inhibitor LCB1 as an example to describe the detailed procedure of hydrazide-based native chemical ligation.