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The simultaneous construction of multiple stereogenic elements in a single step is highly appealing and desirable in the field of asymmetric synthesis. Furthermore, the catalytic enantioselective synthesis of inherently chiral calix[n]arenes with high enantiopurity has long been a challenging endeavor. Herein, we report an enantioselective cobalt-catalyzed C-H activation/annulation for the efficient construction of inherently chiral calix[4]arenes bearing multiple C-N axially chiral element. By employing the benzamide tethered calix[4]arene as the substrate, the C-H annulation with alkynes can be successfully accomplished, leading to the generation of multiple stereogenic elements. A wide range of calix[4]arenes and alkynes are found to be well compatible, and exhibit good yields, high enantioselectivity and excellent diastereoselectivity. Notably, the gram-scale reaction, catalytic application, synthetic transformations, and chiral recognition further showcase the potential applications of this protocol.

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Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) mediated by CD4+ T helper (Th) cells, and characterized by immune cell infiltration, demyelination and neurodegeneration, with no definitive cure available. Thus, it is pivotal and imperative to acquire more profound comprehension of the underlying mechanisms implicated in MS. Dysregulated immune responses are widely believed to play a primary role in the pathogenesis of MS. Recently, a plethora of studies have demonstrated the involvement of T follicular helper (Tfh) cells and tertiary lymphoid-like structures (TLSs) in the pathogenesis and progression of MS. Cathepsin C (CatC) is a cysteine exopeptidase which is crucial for the activation of immune-cell-associated serine proteinases in many inflammatory diseases in peripheral system, such as rheumatoid arthritis and septicemia. We have previously demonstrated that CatC is involved in neuroinflammation and exacerbates demyelination in both cuprizone-induced and experimental autoimmune encephalomyelitis (EAE) mouse models. However, the underlying immunopathological mechanism remains elusive. In the present study, we established a recombinant myelin oligodendrocyte glycoprotein 35-55 peptide-induced EAE model using conditional CatC overexpression mice to investigate the effects of CatC on the alteration of CD4+ Th subsets, including Th1, Th2, Th17, Tfh and T regulatory cells. Our findings demonstrated that CatC particularly enhanced the population of Tfh cell in the brain, resulting in the earlier onset and more severe chronic syndrome of EAE. Furthermore, CatC promoted the formation of TLSs in the brain, leading to persistent neuroinflammation and exacerbating the severity of EAE in the chronic phase. Conversely, treatment with AZD7986, a specific inhibitor of CatC, effectively attenuated the syndrome of EAE and its effects caused by CatC both in vivo and in vitro. These findings provide a novel insight into the critical role of CatC in innate and adaptive immunity in EAE, and specific inhibitor of CatC, AZD7986, may contribute to potential therapeutic strategies for MS.

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Incorporating associative interactions as the energy dissipation units has been recognized as an effective strategy to develop tough hydrogels. For hydrogen-bond associations, however, it is highly challenging to stabilize them under aqueous conditions. Although affording cooperativity can enhance and stabilize the hydrogen bonds, it usually requires stepwise polymerization to form these cooperative associations between different polymers and networks. Here, we report a series of tough supramolecular hydrogels with robust hydrogen-bond associations between grafted polymers that are synthesized by polymerization of a macromonomer of poly(N,N-dimethylacrylamide) (PDMAA) and a small monomer of methacrylic acid. The grafted chains of PDMAA form cooperative hydrogen bonds with the main chain of poly(methacrylic acid) (PMAAc), forming supramolecular hydrogels with high toughness and good stability. The tough and stiff hydrogels are in a glassy state, exhibit forced elastic deformation at room temperature, and remain stable over a wide pH range. In contrast, hydrogels prepared by the copolymerization of DMAA and MAAc are swollen and weak in water due to the lack of successive hydrogen donor/acceptor units and the absence of cooperative hydrogen bonds. In addition, these tough hydrogels exhibit good recyclability and shape memory properties, owing to the supramolecular nature of the network and the temperature-dependent mechanical properties. The influence of polymer structure on the associative interactions and macroscopic properties of the hydrogels should be informative for the design of tough soft materials with versatile applications.

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Here we designed enantiomeric lipid-mimetic glutamic acid derivatives (L/D-UG) and investigated their self-assembled chiral nanostructures and performance with the protein adsorption as well as the osteogenesis. It was found that L or D-UG can self-assemble into vesicle bilayers and two-dimensional (2D) nanocrystals via a kinetic and thermodynamic control, respectively. These chiral vesicles and 2D crystals showed differentiated adsorption of proteins by their curvature and chirality. Specifically, fibronectin constituted by L-amino acids adsorbed preferentially on L-UG 2D crystal in a semi-random pattern and L-2D nanocrystal show as the most effective structures to promote bone regeneration. The controlled vesicle and 2D crystal assemblies with different chirality and curvature helps to clarify their determine roles in protein adsorption and osteogenesis.

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Here, it is shown that photoirradiation triggered chiral J-aggregates formation of an achiral anionic porphyrin, TPPS (tetrakis(4-sulfonatophenyl) porphyrin), in the presence of chiral triphenylamine (TPA) derivatives. A series of chiral triarylamines linked with aromatic rings is designed through urea or amide bonds. UV-irradiation of self-assembled urea-linked triphenylamine derivatives causes the formation of persistent radical cations in the chlorinated solvents, which subsequently induces the aggregation of TPPS. Transferring chirality of TPA derivatives to achiral TPPS J-aggregates leads to the chiral assemblies with remarkable chiroptical signals. The experimental results demonstrate that, TPA derivatives linked by the urea bond can effectively promote the aggregation of TPPS rather than those with the amide bond although the photo-generated radical cations are both produced. It is suggested that the urea-linked TPA derivatives are more favorable to stable radical cations and thus cause the formation of TPPS chiral J-aggregation. This work may open up an avenue for designing photo-modulated chiral supramolecular assemblies.

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Facing the severe problem of microplastic pollution, there is an urgent need to develop biodegradable fibers to replace the petrochemical fibers. Sodium alginate, a biomass polysaccharide, has gained widespread attentions recently for the fiber manufacture. However, the limited mechanical strength of alginate fibers restricts their usages as load-bearing fabrics and reinforcement fibers. Here, we develop a novel strategy to prepare alginate multifilaments using pre-crosslinked sodium alginate solutions. The increase in the pre-crosslinking ratio effectively hinders the disentanglement of sodium alginate chains at high stretches, causing an increase in the shear viscosity of the solution ascertained from the capillarity-driven thinning process from 4.5 Pa·s to 9.9 Pa·s and facilitating the high alignment and orientation of sodium alginate chains. The resultant fibers possess a breaking strength of 474 MPa, elongation at break of 16 %, Young's modulus of 14.4 GPa, and toughness of 51.8 MJ/m3, exceeding most biomass fibers without reinforcement additives. The high orientation degree of 0.865 and high spinnability of alginate multifilaments enable their applications in multi-channel encryption fabrics that exhibit distinct information under various optical conditions. This rheological regulation of spinning solutions provides a facile yet effective strategy to enhance the mechanical performance and broaden application scenarios of alginate fibers.

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Chirality is a fundamental property in nature and is widely observed at hierarchical scales from subatomic, molecular, supramolecular to macroscopic and even galaxy. However, the transmission of chirality across different length scales and the expression of homochiral nano/microstructures remain challenging. Herein, we report the formation of macroscopic homochiral helicoids with ten micrometers from enantiomeric pyromellitic diimide-based molecular triangle (PMDI-Δ) and achiral pyrene via a screw dislocation-driven co-self-assembly. Chiral transfer and expression from molecular and supramolecular levels, to the macroscopic helicoids, is continuous and follows the molecular chirality of PMDI-Δ. Furthermore, the screw dislocation and chirality transfer lead to a unidirectional curvature of the helicoids, which exhibit excellent circularly polarized luminescence with large |glum| values up to 0.05. Our results demonstrate the formation of a homochiral macroscopic organic helicoid and function emergence from small molecules via screw dislocations, which deepens our understanding of chiral transfer and expression across different length scales.

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Extrusion-based 3D printing is a facile technology to construct complex structures of hydrogels, especially for tough hydrogels that have shown demonstrated potential in load-bearing materials and tissue engineering. However, 3D-printed hydrogels often possess mechanical properties that do not guarantee their usage in tissue-mimicking, load-bearing components, and motion sensors. This study proposes a novel strategy to construct high-strength and anisotropic Fe3+ cross-linked poly(acrylamide-co-acrylic acid)/sodium alginate double network hydrogels. The semi-flexible sodium alginate chains act as a "conformation regulator" to promote the formation of strong intermolecular interactions between polymer chains and lock the more extended conformation exerted by the pre-stretch, enabling the construction of 3D-printed hydrogel structures with high orientation. The equilibrated anisotropic hydrogel filaments with a water content of 50-60 wt.% exhibit outstanding mechanical properties (tensile strength: 9-44 MPa; elongation at break: 120-668%; Young's modulus: 7-62 MPa; toughness: 26-52 MJ m- 3). 3D-printed anisotropic hydrogel structures with high mechanical performance show demonstrated potential as loading-bearing structures and electrodes of flexible triboelectric nanogenerators for versatile human motion sensing.

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The attributes of diversity and concealment pose formidable challenges in the accurate detection and efficacious management of distresses within subgrade structures. The onset of subgrade distresses may precipitate structural degradation, thereby amplifying the frequency of traffic incidents and instigating economic ramifications. Accurate and timely detection of subgrade distresses is essential for maintaining and repairing road sections with existing distresses. This helps to prolong the service life of road infrastructure and reduce financial burden. In recent years, the advent of numerous novel technologies and methodologies has propelled significant advancements in subgrade distress detection. Therefore, this review delineates a concentrated examination of subgrade distress detection, methodically consolidating and presenting various techniques while dissecting their respective merits and constraints. By furnishing comprehensive guidance on subgrade distress detection, this review facilitates the expedient identification and targeted treatment of subgrade distresses, thereby fortifying safety and enhancing durability. The pivotal role of this review in bolstering the construction and operational facets of transportation infrastructure is underscored.

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The increasingly severe plastic pollution has urged an inevitable trend to develop biodegradable plastic products that can take over synthetic plastics. As one of the most abundant natural polymers, polysaccharides are an ideal candidate to substitute synthetic plastics. The rigidity of polysaccharide chains principally allows for high strength and stiffness of their materials, however, challenges the facile orientation in material processing. Here, a general hydrogen bond-mediated plasticization strategy to regulate isotropic sodium alginate (SA) chains to a highly ordered state is developed, and alginate plastics with high performances are fabricated. It is revealed that hydroxyl groups in glycerol modulate the viscoelasticity of SA solids by forming strong hydrogen bonds with SA chains, achieving a large stretchability at a high solid content. Highly orientated alginate films exhibit a superior tensile strength of 575 MPa and toughness of 60.7 MJ m-3, outperforming most regenerated biomass films. The high solid content and large stretchability mediated by strong hydrogen bonding ensure plastic molding of solid-like SA with high fidelity. This hydrogen bond-mediated plasticity provides a facile but effective method to justify the high performances of polysaccharide-based plastics.

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A huge production of waste activated sludge (WAS) has been a burden for wastewater treatment plants (WWTPs) with high disposal cost and little benefit back to wastewater purification. The short-chain fatty acids (SCFAs) produced by a short-term acidogenic fermentation of WAS before methane production have been proven to be a high-quality carbon source available for microbial denitrification process. The dual purpose of full recovery of fermentation liquid products and facilitating disposal of residual solid waste necessitate an efficient solid-liquid separation process of short-term fermentation liquid. The transformation and loss of various soluble carbon sources between solid and liquid are very important issues for carbon recovery efficiency when combining short-term fermentation and sludge dewatering in WWTPs. Here we testified the three conventional preconditioning coagulants, Polyferric Sulfate (PFS), Poly Aluminum Chloride (PAC) and Polyacrylamide (PAM), to improve the efficiency of subsequent solid-liquid separation. The results show that conversion yield of SCFAs in the liquid phase of sludge after short-term fermentation was 195 mg COD/g VSS, when using the coagulants PFS, PAC, and PAM for recovery, the recovery ratio was 79.5%, 82.0%, and 85.9%, respectively, while the dewaterability could be improved after preconditioning short-term fermentation sludge. The complexation of Al3+/Fe3+ in metal coagulants with carboxyl groups of SCFA demonstrated by Density Functional Theory calculation led to small part of soluble carbons co-migration to the solid phase, mainly a loss of high molecular weight organic compounds (carbohydrate, proteins, humic acids), while the application of PAM had little impact on carbon recovery. Economic calculations further showed PAM preconditioning short-term fermentation liquid of WAS could achieve higher recovery benefits.

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Although no longer a public health emergency of international concern, COVID-19 remains a persistent and critical health concern. The development of effective antiviral drugs could serve as the ultimate piece of the puzzle to curbing this global crisis. 3-chymotrypsin-like protease (3CLpro), with its substrate specificity mirroring that of the main picornavirus 3C protease and conserved across various coronaviruses, emerges as an ideal candidate for broad-spectrum antiviral drug development. Moreover, it holds the potential as a reliable contingency option to combat emerging SARS-CoV-2 variants. In this light, the approved drugs, promising candidates, and de-novo small molecule therapeutics targeting 3CLpro since the COVID-19 outbreak in 2020 are discussed. Emphasizing the significance of diverse structural characteristics in inhibitors, be they peptidomimetic or nonpeptidic, with a shared mission to minimize the risk of cross-resistance. Moreover, the authors propose an innovative optimization strategy for 3CLpro reversible covalent PROTACs, optimizing pharmacodynamics and pharmacokinetics to better prepare for potential future viral outbreaks.

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While chiral molecular rotors have unique frames and cavities to possibly generate switchable chiroptical functions, it still remains a formidable challenge to precisely restrict their rotations to activate certain functions such as fluorescence as well as circularly polarized luminescence (CPL), which are strongly related to the local molecular rotations. Herein, we design a pair of enantiopure helical cage rotors, which emit light neither at the molecular state nor in the crystal or aggregation states, although they contain luminophore groups. However, upon mounting with fluoroaromatic borane (TFPB) as a molecular brake, the phenyl rotation of the helical cage can be effectively hindered and fluorescence and CPL activities of the molecular cage are switched on. Crystal structure analysis reveals that the rotation is restricted through synergistic B-O-H-N bonding and a fluoroaromatic-aromatic (ArF-Ar) dipole interaction. Moreover, the helical cages are switched on stepwise with color-variable fluorescence and CPL by the inner brake in the molecular state and the outer brake in the supramolecular assemblies, respectively. This work not only provides the design idea of chiroptical molecular rotors but also unveils how fluorescence and CPL could be generated in cage rotor systems.

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Ferroptosis is a newly discovered form of iron-dependent oxidative cell death and drives the loss of neurons in spinal cord injury (SCI). Mitochondrial damage is a critical contributor to neuronal death, while mitochondrial quality control (MQC) is an essential process for maintaining mitochondrial homeostasis to promote neuronal survival. However, the role of MQC in neuronal ferroptosis has not been clearly elucidated. Here, we further demonstrate that neurons primarily suffer from ferroptosis in SCI at the single-cell RNA sequencing level. Mechanistically, disordered MQC aggravates ferroptosis through excessive mitochondrial fission and mitophagy. Furthermore, mesenchymal stem cells (MSCs)-mediated mitochondrial transfer restores neuronal mitochondria pool and inhibits ferroptosis through mitochondrial fusion by intercellular tunneling nanotubes. Collectively, these results not only suggest that neuronal ferroptosis is regulated in an MQC-dependent manner, but also fulfill the molecular mechanism by which MSCs attenuate neuronal ferroptosis at the subcellular organelle level. More importantly, it provides a promising clinical translation strategy based on stem cell-mediated mitochondrial therapy for mitochondria-related central nervous system disorders.

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Non-alcoholic steatohepatitis (NASH), a progressive form of non-alcoholic fatty liver disease (NAFLD), is characterised by chronic liver inflammation, which can further progress into complications such as liver cirrhosis and NASH-associated hepatocellular carcinoma (HCC) and therefore has become a growing health problem worldwide. The type I interferon (IFN) signaling pathway plays a pivotal role in chronic inflammation; however, the molecular mechanisms underlying NAFLD/NASH from the perspective of innate immune response has not yet been fully explored. In this study, we elucidated the mechanisms of how innate immune response modulates NAFLD/NASH pathogenesis, and demonstrated that hepatocyte nuclear factor-1alpha (HNF1A) was suppressed and the type I IFN production pathway was activated in liver tissues of patients with NAFLD/NASH. Further experiments suggested that HNF1A negatively regulates the TBK1-IRF3 signaling pathway by promoting autophagic degradation of phosphorylated-TBK1, which constrains IFN production, thereby inhibiting the activation of type I IFN signaling. Mechanistically, HNF1A interacts with the phagophore membrane protein LC3 through its LIR-docking sites, and mutations of LIRs (LIR2, LIR3, LIR4, and LIRs) block the HNF1A-LC3 interaction. In addition, HNF1A was identified not only as a novel autophagic cargo receptor but also to specifically induce K33-linked ubiquitin chains on TBK1 at Lys670, thereby resulting in autophagic degradation of TBK1. Collectively, our study illustrates the crucial function of the HNF1A-TBK1 signaling axis in NAFLD/NASH pathogenesis via cross-talk between autophagy and innate immunity.

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Background: Polydrug abuse is common among opioid users. Individuals who use both heroin and methamphetamine (MA) have been shown to experience a wide range of cognitive deficits. Previous research shows that repetitive transcranial magnetic stimulation (rTMS) can change cerebral cortical excitability and regulate neurotransmitter concentration, which could improve cognitive function in drug addiction. However, the stimulation time, location, and possible mechanisms of rTMS are uncertain. Methods: 56 patients with polydrug use disorder were randomized to receive 20 sessions of 10 Hz rTMS (n = 19), iTBS (n = 19), or sham iTBS (n = 18) to the left DLPFC. All patients used MA and heroin concurrently. Cognitive function was assessed and several related proteins including EPI, GABA-Aα5, IL-10, etc. were quantified by ELISA before and after the treatment. Results: Baseline RBANS scores were lower than normal for age (77.25; IQR 71.5-85.5). After 20 treatment sessions, in the iTBS group, the RBANS score increased by 11.95 (95% CI 0.02-13.90, p = 0.05). In particular, there were improvements in memory and attention as well as social cognition. Following treatment, serum EPI and GABA-Aα5 were reduced and IL-10 was elevated. The improvement of immediate memory was negatively correlated with GABA-Aα5 (r = -0.646, p = 0.017), and attention was positively correlated with IL-10 (r = 0.610, p = 0.027). In the 10 Hz rTMS group, the improvement of the RBANS total score (80.21 ± 14.08 before vs.84.32 ± 13.80 after) and immediate memory (74.53 ± 16.65 before vs.77.53 ± 17.78 after) was statistically significant compared with the baseline (p < 0.05). However, compared with the iTBS group, the improvement was small and the difference was statistically significant. There was no statistically significant change in the sham group (78.00 ± 12.91 before vs.79.89 ± 10.92 after; p > 0.05). Conclusion: Intermittent theta burst stimulation to the left DLPFC may improve cognitive function in polydrug use disorder patients. Its efficacy appears to be better than that of 10 Hz rTMS. The improvement of cognitive function may be related to GABA-Aα5 and IL-10. Our findings preliminarily demonstrate the clinical value of iTBS to the DLPFC to augment neurocognitive recovery in polydrug use disorders.

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Calcium alginate (CA) hydrogel spheres were widely used as adsorbents to remove organics, but their adsorption capacities and reusability to some antibiotics are unsatisfactory. In this study, calcium alginate/chitosan (CA/CTS) hydrogel spheres were prepared as precursors. Acid-washed CA/CTS (CA/CTS-M) hydrogel spheres (310.6 mg/g) behaved much better adsorption capacity of norfloxacin (NOR) than CA (69.5 mg/g) and CA/CTS (87.7 mg/g) hydrogel spheres. Astonishingly, after being reused for 15 cycles, CA/CTS-M has no loss of NOR adsorption capacity. In the original idea, acid wash was expected to remove the chitosan in CA/CTS hydrogel spheres for obtaining a larger specific surface area. Both scanning electron microscopy and Brunauer-Emmett-Teller test showed that acid wash can remove CTS from CA/CTS hydrogel spheres to increase the specific surface area. However, part of the chitosan remained in CA/CTS-M, having a role to enhance the structural stability of the material, because the acid-washed CA (about 2 mm) has a significantly smaller diameter than CA/CTS-M (about 3 mm). According to the influence of pH and density functional theory calculations, electrostatic attraction is the key driving force of NOR adsorption. Importantly, acid wash led to more negative-charged surface characterized by Zeta potential, which is the main reason of the significantly enhanced adsorption capacity of CA/CTS-M in removal of NOR. In short, CA/CTS-M hydrogel spheres are environment friendly and highly stable adsorbents with high adsorption capacity in the removal of NOR.

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L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA, pepB, pepD, pepN, dpp, and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)-ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, BpADNPA (BPA-ΔpepADN) was built, and the production of Ala-Gln was 129% of the BPA's accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. KEY POINTS: • Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis • The balanced gene knockout combination is pepA, pepD, and pepN • The accumulation of Ala-Gln with BpADNPA was 129% of the control.

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BACKGROUND AND AIMS: Liver transplantation (LT) is the primary curative option for cirrhotic patients with early-stage hepatocellular carcinoma (HCC). However, tumor recurrence occurs in 15-20% of cases with unfavorable prognosis. We have developed a library of T cell receptors (TCRs) specific for different hepatitis B virus (HBV) antigens, restricted by different molecules of human leucocyte antigen (HLA)-class I, to redirect T cells against HBV antigens (Banu in Sci Rep 4:4166, 2014). We further demonstrated that these transiently functional T cells specific for HBV obtained through messenger RNA (mRNA) electroporation can eliminate HCC cells expressing HBV antigens in vitro and in vivo (Kah in J Clin Invest 127:3177-3188, 2017). A phase I clinical trial for patients with HCC recurrence post-liver transplant was conducted to assess the safety, tolerability, and anti-tumor efficacy of transiently functional HBV-TCR T cells. Here, we report the clinical findings with regard to the safety and anti-tumor efficacy of mRNA electroporated HBV-specific TCR-T cells. (ClinicalTrials.gov identifier: NCT02719782). PATIENTS AND METHODS: A total of six patients with HBV-positive recurrent HCC post-liver transplant and HLA-matched to TCR targeting hepatitis B surface antigen (HBsAg) or hepatitis B core antigen (HBcAg) (HLA-A*02:01/HBsAg, HLA-A*11:01/HBcAg, HLA-B*58:01/HBsAg or HLA-C*08:01/HBsAg) were enrolled in this study. The primary objective was to assess the safety of short-lived mRNA electroporated HBV-TCR T cells based on the incidence and severity of the adverse event (AE) graded per National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE), Version 4.0. The secondary objective was to determine the effectiveness of HBV-TCR T cells as per RECIST 1.1 criteria. Patients were followed up for survival for 2 years post-end of treatment. RESULTS: The median age of the six patients was 35.5 years (range: 28-47). The median number of HBV-TCR T cell infusions administered was 6.5 (range: 4-12). The treatment-related AE included grade 1 pyrexia. This study reported no cytokine release syndrome nor neurotoxicity. One patient remained alive and five were deceased at the time of the data cutoff (30 April 2020). CONCLUSION: This study has demonstrated that multiple infusions of mRNA electroporated HBV-specific TCR T cells were well-tolerated in patients with HBV-positive recurrent HCC post-liver transplant.

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l-Alanyl-l-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the N-glycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g·(L·min)-1, respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.

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