Structure-Guided Development of Bivalent Aptamers Blocking SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused devastation to human society through its high virulence, infectivity, and genomic mutations, which reduced the efficacy of vaccines. Here, we report the development of aptamers that effectively interfere with SARS-CoV-2 infection by targeting its spike protein, which plays a pivotal role in host cell entry of the virus through interaction with the viral receptor angiotensin-converting enzyme 2 (ACE2). To develop highly effective aptamers and to understand their mechanism in inhibiting viral infection, we determined the three-dimensional (3D) structures of aptamer/receptor-binding domain (RBD) complexes using cryogenic electron microscopy (cryo-EM). Moreover, we developed bivalent aptamers targeting two distinct regions of the RBD in the spike protein that directly interact with ACE2. One aptamer interferes with the binding of ACE2 by blocking the ACE2-binding site in RBD, and the other aptamer allosterically inhibits ACE2 by binding to a distinct face of RBD. Using the 3D structures of aptamer-RBD complexes, we minimized and optimized these aptamers. By combining the optimized aptamers, we developed a bivalent aptamer that showed a stronger inhibitory effect on virus infection than the component aptamers. This study confirms that the structure-based aptamer-design approach has a high potential in developing antiviral drugs against SARS-CoV-2 and other viruses.

Novel bispecific human antibody platform specifically targeting a fully open spike conformation potently neutralizes multiple SARS-CoV-2 variants.

Rapid emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted an urgent need for the development of broadly applicable and potently neutralizing antibody platform against the SARS-CoV-2, which can be used for combatting the coronavirus disease 2019 (COVID-19). In this study, based on a noncompeting pair of phage display-derived human monoclonal antibodies (mAbs) specific to the receptor-binding domain (RBD) of SARS-CoV-2 isolated from human synthetic antibody library, we generated K202.B, a novel engineered bispecific antibody with an immunoglobulin G4-single-chain variable fragment design, with sub- or low nanomolar antigen-binding avidity. Compared with the parental mAbs or mAb cocktail, the K202.B antibody showed superior neutralizing potential against a variety of SARS-CoV-2 variants in vitro. Furthermore, structural analysis of bispecific antibody-antigen complexes using cryo-electron microscopy revealed the mode of action of K202.B complexed with a fully open three-RBD-up conformation of SARS-CoV-2 trimeric spike proteins by simultaneously interconnecting two independent epitopes of the SARS-CoV-2 RBD via inter-protomer interactions. Intravenous monotherapy using K202.B exhibited potent neutralizing activity in SARS-CoV-2 wild-type- and B.1.617.2 variant-infected mouse models, without significant toxicity in vivo. The results indicate that this novel approach of development of immunoglobulin G4-based bispecific antibody from an established human recombinant antibody library is likely to be an effective strategy for the rapid development of bispecific antibodies, and timely management against fast-evolving SARS-CoV-2 variants.

Synthesis and drug release properties of melanin added functional allopurinol incorporated starch-based biomaterials.

The main objective of this study was to prepare functional allopurinol (ALP) incorporated biomaterials using mungbean starch, polyvinyl alcohol, melanin (MEL), and plasticizers. Prepared biomaterials were characterized by FE-SEM and FT-IR analysis. Photothermal conversion efficiencies and ALP release properties of biomaterials were evaluated with NIR laser irradiation. When biomaterials were irradiated with the NIR laser, temperatures increase of MEL-added biomaterials were higher than those of MEL-non-added biomaterials. After NIR laser irradiation, ALP release rates of MEL-added biomaterials were 1.62 times faster than those of MEL-non-added biomaterials. In addition, ALP release using an artificial skin was increased by NIR laser irradiation. ALP release from biomaterials followed Fickian diffusion mechanism, while ALP release using an artificial skin followed a non-Fickian diffusion mechanism. Xanthine oxidase inhibitory (%) for MEL-added biomaterials with/without the addition of GL and XL were 47.5%, 61.7%, and 65.1%, respectively.

Preparation and evaluation of functional allopurinol imprinted starch based biomaterials for transdermal drug delivery.

This study focuses on the synthesis of functional allopurinol (ALP) imprinted biomaterials for a transdermal drug delivery using mung bean starch (MBS), polyvinyl alcohol (PVA), sodium benzoate (SB) as a crosslinking agent, and poloxamer (PX) as a thermo-sensitive polymer. Prepared functional biomaterials were characterized and evaluated by SEM, FT-IR analysis, and physical properties. Results of ALP recognition properties indicated that adsorbed amounts (Q) of ALP on functional ALP imprinted biomaterials were 3.8 to 4.9-fold higher than that of non-ALP imprinted biomaterial. Results of ALP release revealed that the ALP release rate for PX added biomaterials was 1.10 (36.5 °C) or 1.30 (45 °C) times faster than that at 25 °C. These results indicate that functional ALP imprinted biomaterials have thermo-sensitive properties due to the addition of PX. Results of ALP release using artificial skin indicated that ALP release was increased at a relatively steady-state rate for 3 h and that the ALP release behavior followed the non-Fickian diffusion mechanism.

The Effects of Service Employee Resilience on Emotional Labor: Double-Mediation of Person-Job Fit and Work Engagement.

This study examined the effect of service employees' resilience on deep acting in the job demands-resources model (JD-R model). It set and verified person-job fit and work engagement as double-mediation factors between service employees' resilience and deep acting. To accomplish this, surveys targeting service employees working in the retail finance industry in Korea were administered. The analysis showed that resilience significantly increased person-job fit, and person-job fit improved work engagement. Additionally, it showed that work engagement improved deep acting. With regard to the double-mediation effect, the direct effect of resilience on deep acting was not statistically significant, but the double-mediation effect through person-job fit and work engagement was significant. In other words, person-job fit and work engagement fully mediated the relationship between resilience and deep acting. Additionally, person-job fit alone did not mediate the relationship between resilience and deep acting, but the independent mediation effect of work engagement was significant.

Phosphorylation of OCT4 Serine 236 Inhibits Germ Cell Tumor Growth by Inducing Differentiation.

Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

Preparation and release properties of arbutin imprinted inulin/polyvinyl alcohol biomaterials.

The main objective of this work was to prepare inulin (INL)/polyvinyl alcohol (PVA) biomaterials imprinted with arbutin (AR) as the target drug. INL from Jerusalem artichoke flour was extracted with hot water extraction method. INL/PVA biomaterials were synthesized with a casting method and a UV curing. The optimal UV curing time and sodium benzoate content were about 10 min and 0.1 wt%, respectively. The biomaterials were characterized by SEM and FT-IR analysis. Mechanical properties of prepared AR imprinted biomaterials were also investigated. AR release was examined with changes of pH at 36.5 °C. The AR release ratio was also investigated using artificial skin. It was found that AR was released constantly for 40 min. Results of drug release mechanism indicated that AR release followed the Fickian diffusion behavior, whereas drug release using artificial skin followed the non-Fickian diffusion behavior. Tyrosinase inhibitory (%) for AR imprinted biomaterials with/without the addition of GL were 58.8% and 79.2%, respectively.

Publisher Correction: Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A case of successful late steroid withdrawal after ABO-incompatible kidney transplantation.

Few data exist regarding steroid withdrawal in ABO-incompatible (ABO-i) kidney transplantation (KT). Here, we report a case of steroid withdrawal after ABO-i KT. A 46-year-old man diagnosed with Henoch-Schonlein purpura received ABO-i KT from his 42-year-old sister. The recipient and donor blood types were O and AB, respectively. His preoperative ABO antibody titers were anti-A of 1:16 and anti-B of 1:8 in isoagglutinin test. HLA mismatch was 0 and he received a single 325 mg/m2 dose of intravenous (IV) rituximab 4 weeks before KT. Three sessions of plasma exchange were undertaken before KT and low-dose IV immunoglobulin of 0.1 g/kg was administered after plasma exchange. On the day of the operation, ABO antibody titer decreased to anti-A of 1:4 and anti-B of 1:2. Renal function remained stable after KT. The patient wished to stop steroid treatment despite the risk of rejection after withdrawal. Steroid tapering was initiated at 20 months and accomplished at 26 months after KT. At that time, serum creatinine level was 1.13 mg/dL, and anti-A and anti-B titers were 1:8 and 1:2, respectively. No issues were observed after steroid withdrawal. At 48 months after KT, serum creatinine level was 1.21 mg/dL, and anti-A and anti-B antibody titers were 1:32 and 1:2, respectively. Steroid withdrawal in ABO-i KT might be considered in immunologically low-risk patients.

Cryo-EM structure of the human MLL1 core complex bound to the nucleosome.

Mixed lineage leukemia (MLL) family histone methyltransferases are enzymes that deposit histone H3 Lys4 (K4) mono-/di-/tri-methylation and regulate gene expression in mammals. Despite extensive structural and biochemical studies, the molecular mechanisms whereby the MLL complexes recognize histone H3K4 within nucleosome core particles (NCPs) remain unclear. Here we report the single-particle cryo-electron microscopy (cryo-EM) structure of the NCP-bound human MLL1 core complex. We show that the MLL1 core complex anchors to the NCP via the conserved RbBP5 and ASH2L, which interact extensively with nucleosomal DNA and the surface close to the N-terminal tail of histone H4. Concurrent interactions of RbBP5 and ASH2L with the NCP uniquely align the catalytic MLL1SET domain at the nucleosome dyad, thereby facilitating symmetrical access to both H3K4 substrates within the NCP. Our study sheds light on how the MLL1 complex engages chromatin and how chromatin binding promotes MLL1 tri-methylation activity.

MMOD-induced structural changes of hydroxylase in soluble methane monooxygenase.

Soluble methane monooxygenase in methanotrophs converts methane to methanol under ambient conditions. The maximum catalytic activity of hydroxylase (MMOH) is achieved through the interplay of its regulatory protein (MMOB) and reductase. An additional auxiliary protein, MMOD, functions as an inhibitor of MMOH; however, its inhibitory mechanism remains unknown. Here, we report the crystal structure of the MMOH-MMOD complex from Methylosinus sporium strain 5 (2.6 Å). Its structure illustrates that MMOD associates with the canyon region of MMOH where MMOB binds. Although MMOD and MMOB recognize the same binding site, each binding component triggers different conformational changes toward MMOH, which then respectively lead to the inhibition and activation of MMOH. Particularly, MMOD binding perturbs the di-iron geometry by inducing two major MMOH conformational changes, i.e., MMOH ß subunit disorganization and subsequent His147 dissociation with Fe1 coordination. Furthermore, 1,6-hexanediol, a mimic of the products of sMMO, reveals the substrate access route.

A Catalytic Trisulfide in Human Sulfide Quinone Oxidoreductase Catalyzes Coenzyme A Persulfide Synthesis and Inhibits Butyrate Oxidation.

Mitochondrial sulfide quinone oxidoreductase (SQR) catalyzes the oxidation of H2S to glutathione persulfide with concomitant reduction of CoQ10. We report herein that the promiscuous activity of human SQR supported the conversion of CoA to CoA-SSH (CoA-persulfide), a potent inhibitor of butyryl-CoA dehydrogenase, and revealed a molecular link between sulfide and butyrate metabolism, which are known to interact. Three different CoQ1-bound crystal structures furnished insights into how diverse substrates access human SQR, and provided snapshots of the reaction coordinate. Unexpectedly, the active site cysteines in SQR are configured in a bridging trisulfide at the start and end of the catalytic cycle, and the presence of sulfane sulfur was confirmed biochemically. Importantly, our study leads to a mechanistic proposal for human SQR in which sulfide addition to the trisulfide cofactor eliminates 201Cys-SSH, forming an intense charge-transfer complex with flavin adenine dinucleotide, and 379Cys-SSH, which transfers sulfur to an external acceptor.

Monitoring circulating tumor DNA by analyzing personalized cancer-specific rearrangements to detect recurrence in gastric cancer.

Circulating tumor DNA (ctDNA) has emerged as a candidate biomarker for cancer screening. However, studies on the usefulness of ctDNA for postoperative recurrence monitoring are limited. The present study monitored ctDNA in postoperative blood by employing cancer-specific rearrangements. Personalized cancer-specific rearrangements in 25 gastric cancers were analyzed by whole-genome sequencing (WGS) and were employed for ctDNA monitoring with blood up to 12 months after surgery. Personalized cancer-specific rearrangements were identified in 19 samples. The median lead time, which is the median duration between a positive ctDNA detection and recurrence, was 4.05 months. The presence of postoperative ctDNA prior to clinical recurrence was significantly correlated with cancer recurrence within 12 months of surgery (P = 0.029); in contrast, no correlation was found between cancer recurrence and the presence of preoperative ctDNA, suggesting the clinical usefulness of postoperative ctDNA monitoring for cancer recurrence in gastric cancer patients. However, the clinical application of ctDNA can be limited by the presence of ctDNA non-shedders (42.1%, 8/19) and by inconsistent postoperative ctDNA positivity.

S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase-dependent hydrogen sulfide synthesis.

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 µm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine ß-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

Electrical Heating Performance of Electro-Conductive Para-aramid Knit Manufactured by Dip-Coating in a Graphene/Waterborne Polyurethane Composite.

An electro-conductive para-aramid knit was manufactured by a dip-coating in a graphene/waterborne polyurethane(WPU) composite for confirming to use as a fabric heating element applicable to a protective clothing requiring durability. The para-aramid knit was dipped in 8 wt% graphene/WPU composite solution up to five-coat cycles. As a result of electro-conductive textile by number of dip-coating cycles, the electrical, and specifically electrical heating performances were increased number of cycles from one to five. The sample with the best electrical and electrical heating performance was the five-coat sample, and to improve those properties it was hot-pressed at 100 °C, 120 °C, 140 °C and 160 °C. After hot pressing, the entire surface of the sample was filled with graphene/WPU composite and indicated smoothly surface, thus the electrical and electrical heating performance was improved than the five-coat sample. The best performance of was indicated hot-pressed at 140 °C, with a surface resistivity and capacitance of 7.5 × 104 Ω/sq and 89.4 pF, respectively. When a voltage of 50 V was applied, the surface temperature reached 54.8 °C. The five-coat sample with hot-pressed at 140 °C could be applied to a heat-resistant para-aramid knit glove with the touch screen of a mobile phone and electric heating performance.

Primary renal well-differentiated neuroendocrine tumors: report of six cases with an emphasis on the Ki-67 index and mitosis.

BACKGROUND: Primary renal well-differentiated neuroendocrine tumors (WDNETs) also called carcinoid and atypical carcinoid are extremely rare, and little is known about parameters that may predict prognosis at diagnosis. METHODS: Six cases of primary renal WDNET were collected. After reviewing slides stained with hematoxylin and eosin, proportions of each growth pattern were determined. Synaptophysin, chromogranin, CD56, and Ki-67 immunostaining and Ki-67 morphometric analysis were performed. RESULTS: Patients included three female and three males, mean age was 53.3 years. The mean tumor size was 4.5 cm, three cases were greater than 5 cm. At the time of initial surgery, lymph node and/or distant metastasis was confirmed in two cases. In a third case, no metastasis was initially found, but lymph node metastasis was identified during follow-up. The remaining three cases did not exhibit metastasis. Histopathologically, the renal WDNETs were primarily composed of ribbon-like and sheet-like growth patterns. Most of the tumors were diffusely positive for neuroendocrine markers. Mitotic count was high (≥2/10HPF) in cases with lymph node or distant metastasis but was low (< 2/10HPF) in non-metastatic cases. Furthermore, the Ki-67 index was also higher (≥3%) in the cases with metastases than in cases without metastasis. CONCLUSION: Three out of the six primary renal WDNETs demonstrated aggressive behavior and exhibited increased mitotic counts and Ki-67 indices. These results suggest that mitosis and the Ki-67 index could be used as prognostic indicators for renal WDNET.

Endoscopic ultrasound-guided fine needle aspiration with liquid-based cytology preparation in the diagnosis of metastatic small-cell carcinoma in the pancreas.

Pancreatic metastasis is extremely rare, particularly from small-cell lung cancer (SCLC). Studies on the role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) with liquid-based cytology (LBC) in the diagnosis of metastatic small-cell carcinoma in the pancreas have been rarely conducted. We report herein a case of pancreatic metastasis from SCLC diagnosed using EUS-FNA with LBC (ThinPrep). A 71-year-old man presented with chief complaints of hemoptysis and jaundice over the past 1 month. Lung & pancreas tumors with multiple liver nodules were detected on computed tomography. The aspirated material from the pancreas using EUS-FNA was prepared as a cytologic specimen with ThinPrep method, which revealed scattered and clustered "small blue cells" with scant cytoplasm and stippled chromatin with frequent apoptotic bodies. Immunocytochemical staining of the cellblock material revealed strong positivity for CD56 and thyroid transcription factor-1. Endobronchial biopsy for lung mass revealed nests of small, round, blue tumor cells with hyperchromatic nuclei showing salt and pepper chromatin, scant cytoplasm, and brisk mitotic activity. Therefore, a diagnosis of metastatic small-cell carcinoma to the pancreas with an extensive stage was finally made.

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks.

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Structure-based nuclear import mechanism of histones H3 and H4 mediated by Kap123.

Kap123, a major karyopherin protein of budding yeast, recognizes the nuclear localization signals (NLSs) of cytoplasmic histones H3 and H4 and translocates them into the nucleus during DNA replication. Mechanistic questions include H3- and H4-NLS redundancy toward Kap123 and the role of the conserved diacetylation of cytoplasmic H4 (K5ac and K12ac) in Kap123-mediated histone nuclear translocation. Here, we report crystal structures of full-length Kluyveromyces lactis Kap123 alone and in complex with H3- and H4-NLSs. Structures reveal the unique feature of Kap123 that possesses two discrete lysine-binding pockets for NLS recognition. Structural comparison illustrates that H3- and H4-NLSs share at least one of two lysine-binding pockets, suggesting that H3- and H4-NLSs are mutually exclusive. Additionally, acetylation of key lysine residues at NLS, particularly H4-NLS diacetylation, weakens the interaction with Kap123. These data support that cytoplasmic histone H4 diacetylation weakens the Kap123-H4-NLS interaction thereby facilitating histone Kap123-H3-dependent H3:H4/Asf1 complex nuclear translocation.

CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer.

BACKGROUND: Recently, we reported cytoskeleton-associated protein2 (CKAP2) as a possible new prognostic breast cancer marker. However, it has not yet been applied in clinic. Therefore, clinical significance of CKAP2 was evaluated in comparison with that of Ki-67 in a cohort of breast cancer patients, and the expression difference was analyzed in cell cycle-arrested cancer and fibroblast cells. METHODS: A total of 579 early breast cancer patients who underwent surgery at the National Cancer Center Hospital in Korea between 2001 and 2005 were accrued. CKAP2-positive cell count (CPCC) and Ki-67 labeling index (Ki-67LI) were evaluated by immunohistochemcal staining. The immunocytochemical staining patterns of CKAP2 and Ki-67 were analyzed in HeLa and human fibroblast cells after synchronization by double thymidine block. RESULTS: Although there was a significant correlation (R = 0.754, P < 0.001) between CPCC and Ki-67LI, only CPCC was correlated with DFS in overall population (HR, 2.029; 95% CI, 1.012-4.068; P = 0.046) and HER2-negative luminal subgroup (HR, 3.984; 95% CI, 1.350-11.762; P = 0.012) by multivariate analysis. In immunocytochemical staining, more than 50% of serum-starved or non-mitotic cell phase HeLa cells were positive for Ki-67, in comparison to the low CKAP2-positivity, which might explain the prognostic difference between CPCC and Ki-67LI. CONCLUSIONS: The current study showed that CPCC but not Ki-67LI is an independent prognostic indicator in early breast cancer, more specifically in HER2-negative luminal breast cancer. The difference between two markers may be related to the lower background expression of CKAP2 in cancer cells.