An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1-Nb2, with tight affinity and super wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1-Nb2 has a strong escape resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1-Nb2 broadly neutralizes multiple SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1) and Mu (B.1.621). Furthermore, a heavy chain antibody is constructed by fusing the human IgG1 Fc to Nb1-Nb2 (designated as Nb1-Nb2-Fc) to improve its neutralization potency, yield, stability and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1-Nb2-Fc keeps a firm affinity (KD < 1.0x10-12 M) and strong neutralizing activity (IC50 = 0.0017 nM). Together, we developed a tetravalent biparatopic human heavy chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

Inhibition of SAR S-CoV-2 infection and replication by lactoferrin, MUC1 and α-lactalbumin identified in human breastmilk

The global pandemic of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection confers great threat to the public health. Human breastmilk is an extremely complex with nutritional composition to nourish infants and protect them from different kinds of infection diseases and also SARS-CoV-2 infection. Previous studies have found that breastmilk exhibited potent antiviral activity against SARS-CoV-2 infection. However, it is still unknown which component(s) in the breastmilk is responsible for its antiviral activity. Here, we identified Lactoferrin (LF), MUC1 and -Lactalbumin (-LA) from human breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and in vitro confirmation that inhibited SARS-CoV-2 infection and analyzed their antiviral activity using the SARS-CoV-2 pseudovirus system and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP) in the Huh7.5, Vero E6 and Caco-2-N cell lines. Additionally, we found that LF and MUC1 could inhibit viral attachment, entry and post-entry replication, while -LA just inhibit viral attachment and entry. Importantly, LF, MUC1 and -LA possess potent antiviral activities towards not only wild-type but also variants such as B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.1 (kappa). Moreover, LF from other species (e.g., bovine and goat) is still capable of blocking viral attachment to cellular heparan sulfate. Taken together, our study provided the first line of evidence that human breastmilk components (LF, MUC1 and -LA) are promising therapeutic candidates warranting further development or treatingVID-19 given their exceedingly safety levels.

Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

Recently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations {Delta}145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, {Delta}145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

Mutation Y453F in the spike protein of SARS-CoV-2 enhances interaction with the mink ACE2 receptor for host adaption

COVID-19 patients transmitted SARS-CoV-2 to minks in the Netherlands in April 2020. Subsequently, the mink-associated virus (miSARS-CoV-2) spilled back over into humans. Genetic sequences of the miSARS-CoV-2 identified a new genetic variant known as "Cluster 5" that contained mutations in the spike protein. However, the functional properties of these "Cluster 5" mutations have not been well established. In this study, we found that the Y453F mutation located in the RBD domain of miSARS-CoV-2 is an adaptive mutation that enhances binding to mink ACE2 and other orthologs of Mustela species without compromising, and even enhancing, its ability to utilize human ACE2 as a receptor for entry. Structural analysis suggested that despite the similarity in the overall binding mode of SARS-CoV-2 RBD to human and mink ACE2, Y34 of mink ACE2 was better suited to interact with a Phe rather than a Tyr at position 453 of the viral RBD due to less steric clash and tighter hydrophobic-driven interaction. Additionally, the Y453F spike exhibited resistance to convalescent serum, posing a risk for vaccine development. Thus, our study suggests that since the initial transmission from humans, SARS-CoV-2 evolved to adapt to the mink host, leading to widespread circulation among minks while still retaining its ability to efficiently utilize human ACE2 for entry, thus allowing for transmission of the miSARS-CoV-2 back into humans. These findings underscore the importance of active surveillance of SARS-CoV-2 evolution in Mustela species and other susceptible hosts in order to prevent future outbreaks.

Treatment progress of primary light-chain amyloidosis / 白血病·淋巴瘤

Primary light-chain (AL) amyloidosis is a rare, fatal and clonal plasma cell dyscrasia. The bortezomib-based treatment has improved the survival of AL amyloidosis patients, but the prognosis of advanced patients is still poor. More effective novel agents are urgently needed. At the 62nd American Society of Hematology (ASH) Annual Meeting, the clinical data of a variety of new drugs (such as anti-CD38 monoclonal antibody, bcl-2 inhibitor combined with statins and CAEL-101, etc.) were reported.

Progress in pharmacogenomics and efficacy prediction of levetiracetam / 中华神经科杂志

Levetiracetam (LEV) is the second generation of broad-spectrum antiepileptic drugs. Compared with other antiepileptic drugs, LEV has unique antiepileptic mechanism, good efficacy and tolerance, and its target is synaptic vesicle protein 2A. With the widespread use of LEV, more and more adverse reactions have been reported, especially mental related adverse reactions. This paper reviewed the research progress of LEV pharmacogenomics related targets, metabolism, adverse reaction related genetic variation and efficacy prediction, so as to provide decision-making for the application of LEV individualized treatment in clinical practice, improve the quality of life of epileptic patients and reduce the disease burden of patients with epilepsy.

Chemical constituents of Daphne odora var. margirmta / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Daphne odora var. margirmta.</p><p><b>METHOD</b>The compounds were isolated and purified by column chromatography of silica gel, ODS, Sephadex LH-20, and their structures were identified by UV, 1H-NMR, 13C-NMR, MS and CD spectroscopic methods.</p><p><b>RESULT</b>Ten compounds were elucidated as wikatrols B (1), kaempferol (2), daphnodorin B (3), daphnodorin D2 (4), daphnodorin A (5), daphnodorin C (6), daphnodorin D1 (7), daphnodorin I (8), daphnetin (9), daphnoretin (10).</p><p><b>CONCLUSION</b>Compounds 1,2,4 were obtained from this plant for the first time,and compound 1 was isolated firstly from the genus Daphne.</p>

Adsorption Property of Supported Ionic Liquids for Flavonoids / 分析化学

The effects of adsorption time, solid-liquid ratio, sample concentration, flow rate on the adsorption properties of supported ionic liquids(the N-methylimidazolium functionalized silica, SilprMin) for flavonoids were investigated. The results indicated that the adsorption equilibrium for tested compounds, genistein, luteolin and quercetin, was achieved within 30 min, the adsorption efficiencies of these three compounds were improved with the increase of solid-liquid ratio, and decreased with the increase of their concentrations. Moreover, the adsorption isotherm data of the three tested compounds were in good concordance with the Langmuir model. The saturated adsorption capacity of SilprMim for genistein, luteolin and quercetin was 47.7, 52.5 and 63.2 mg/g, respectively. Adsorption efficiency of SilprMim could reach more than 90% at a sample flow rate of 0.5～1.5 mL/min. Using methanol as eluent, the saturated desorption efficiencies of genistein, luteolin and quercetin were 86.1%, 83.3% and 84.6%, respectively. The elution order of the three tested flavonoids was genistein, luteolin and quercetin. SilprMim had strong adsorption and separation capacity for three tested flavonoids, which was hopeful to be applied in separation and purification of naturally occurring flavonoids.

The clinical application of leukocyte VCS parameters in the detection of blood bacterial infection / 中华检验医学杂志

Objective To investigate the clinical application of VCS parameters of leukocyte in the detection of blood bacterial infection, Methods The subjects consisted of 120 patients with blood bacterial infection,69 non-infectious fever patients and 67 health controls.The VCS parameters of neutrophil and lymphocyte were examined with Coulter LH 750 hematology analyzer.The parameters examined including mean channel of neutrophil volume(MNV),neutrophil volume distribution width (NDW) ,mean channel of neutrophil conductivity (MNC),mean channel of neutrophil scatter (MNS),mean channel of lymphocyte volme(MLV),lymphocyte volume distfibufion width (LDW),mean channel of lymphocyte conductivity (MLC) and mean channel of lymphocyte scatter (MLS).Additionally,120 blood bacterial infection patients were grouped according to WBC count(WBC≤10×109/L group and WBC＞10×109/L group),neutrophii rate(≥85%group and＜85%group)and bacterial stain(Gram positive bacteria group and Gram negative bacteria group).VCS parameters among these groups were compared.Results The results of blood infection group were as follows:MNV 156±15,NDW 23.31±3.72,MNS 137±7,MLV 87±12,LDW 17.50±3.38.MLC 110±5 and MLS 69±12.The results of non-infectious fever group were as follows:MNV 151±8,NDW 21.33 ±2.62,MNS 132±10,MLV 91±4.LDW 15.78±1.96.MLC 117±4 and MLS 62±6.The results of control group were as follows:MNV145 ±5.NDW 18.43±0.93.MNS 143 ±4,MLV 84±2,LDW 13.30±0.76.MLC 108±1 and MLS 62±2.There were significant diffierences among these three groups (F value were 19.295,26.272,32.767,6.226,31.016,23.739 and 12.662 respectively,P＜0.05 or P＜0.01).In the infection group.the MNV and NDW were 152 ±16 and 22.19±3.45 respectively for WBC≤10×109/L group.159±12 and 25.29±3.43 respectively for WBC＞10×109/L group.They were both significantly different compared with control group (F valRe were 21.575 and 40.856 respectively,P＜0.01).Also in the infection group.the MNV and NDW were 159±12 and 24.88 ±3.74 respectively for neutrophil rate≥85%group.151±16 and 21.68±2.29 respectively for neutrophil rate＜85%group.They were both significantly different compared with control group(F value were 23.76 and 43.22 respectively,P＜0.01).The MNV and NDW were 157±15 and 24.25±3.39 respectively in those cases with gram-negative bacteremia,153±14 and 21.51±3.78 respectively in those cases with gram-positive bacteremia.They were both signifieanfly difierent compared with control group (F value were 18.74 and 37.47 respectively,P＜0.01).With a cut-off value of 20.50 for the NDW,a sensitivity of 76.7%and specificity of 98.3% were achieyed in diagnosing blood hacterial infection.Conclusion The VCS parameters can reflect the morphologic change of leukocyte in blood bacterial infection.Additionally.the NDW can detect blood bacterial infection more sensitively and specifically.

EFFECT OF SULFORAPHANE ON PROLIFERATION, CELL CYCLE AND APOPTOSIS OF BREAST CANCER CELL LINES AND ITS MECHANISM / 营养学报

Objective: To study the effect of sulforaphane (SUL) on cell growth inhibition, cell cycle, apoptosis and its mechanism in different breast cancer cell lines. Methods: By means of MTT assay, flow cytometry and Western blotting, the effects of the SUL different concentrations on cell growth inhibition, cell cycle arrest, F3Ⅱapoptosis and expression of p34cdc2 and Cdc25C were studied. Results: (1) SUL had strong inhibition effects on the cell growth of tested mammary cancer cell lines, in which the sensitivity of ERP cell lines to SUL was stronger than that of ERN cell line. (2) SUL exhibited obviously G2/M cell cycle arrest to F3Ⅱ and two kinds of ERP cell lines at 5-10?mol/L, whereas no effect on the cell cycle of ERN cell line. (3)The mechanism of hindering transition of F3 Ⅱ cells from G2 phase to M phase was enhancing the phosphorylation of Cdc2, down-regulating the expression of Cdc25C, and inducing inhibition to thedephosphorylation of cyclin B1-Cdc2 complex. (4) No apoptosis was observed under tested conditions. Conclusion: Sulforaphane exhibited obvious differences in the inhibiting effects on the cell proliferation and cell cycle arrests of four different tested breast cancer cell lines, and did not induce apoptosis in F3Ⅱcell line under tested conditions. The mechanism of cell cycle arrest of F3Ⅱcell line appears to involve enhancing the phosphorylation of Cdc2, and down-regulating the expression of Cdc25C.

Process of three-effect dynamic countercurrent extraction from Hedyotis diffusa / 中草药

Objective To study the lab-scale simulated method of three-effect dynamic countercurrent extraction from Hedyotis diffusa by multi-index optimization and SPSS software.Methods Polyphenol content,flavonoids content,scavenging activity of samples on DPPH radicals,and extracting rate were considered as the optimization indexes.Orthogonal design method was used to optimize the four main factors of alcohol concentration,ratio of solid to solution,extracting temperature,and extracting times.Results The optimum condition is 70 min extraction time,solid to solution ratio of 1∶14,50% alcohol,extracting temperature 85 ℃.Conclusion The condition of large-scale industrialized production might be explored by studying the lab-scale simulated method of dynamic countercurrent extraction from H.diffusa.