Development of a bispecific nanobody conjugate broadly neutralizes diverse SARS-CoV-2 variants and structural basis for its broad neutralization.

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future.

Simvastatin encapsulated in exosomes can enhance its inhibition of relapse after orthodontic tooth movement.

INTRODUCTION: Relapse after orthodontic treatment is a major clinical issue in the dental field. Studies indicate that simvastatin may, to some extent, decrease the rate and magnitude of relapse status. Recent evidence demonstrated that exosome-based drug delivery has a broad prospect of clinical application. Hence, this study investigates whether simvastatin encapsulated in exosomes can inhibit relapse after orthodontic tooth movement (OTM). METHODS: Periodontal ligament stem cells (PDLSCs) and their exosomes (PDLSCs-Exo) were isolated and identified. Exosomal simvastatin was obtained by co-incubation of simvastatin and PDLSCs-Exo. An OTM rat model was established. During the relapse period, rats' local alveolar bone was injected with simvastatin, PDLSCs-Exo, and exosomal simvastatin to examine the effect on relapse. Finally, we analyzed the influence of exosomal simvastatin on osteogenesis at the molecular and histologic levels. RESULTS: PDLSCs and PDLSCs-Exo were successfully extracted and characterized by multiple means. Simvastatin encapsulated in exosomes can increase the solubility of the drug. Exosomal simvastatin can enhance its inhibition of relapse after OTM in the rat model. The expression level of osteogenic-related genes and proteins in the exosomal simvastatin group is higher than in other groups. Histologic analysis showed a reduction of bone-resorptive lacunae in the exosomal simvastatin group. CONCLUSIONS: Encapsulating simvastatin into the exosomes derived from PDLSCs can improve simvastatin solubility and enhance the inhibition effect of relapse in the rat model of OTM. Notably, local injection of PDLSCs-Exo alone can also block the relapse after OTM.

COVID-19: data-driven dynamics, statistical and distributed delay models, and observations.

COVID-19 was declared as a pandemic by the World Health Organization on March 11, 2020. Here, the dynamics of this epidemic is studied by using a generalized logistic function model and extended compartmental models with and without delays. For a chosen population, it is shown as to how forecasting may be done on the spreading of the infection by using a generalized logistic function model, which can be interpreted as a basic compartmental model. In an extended compartmental model, which is a modified form of the SEIQR model, the population is divided into susceptible, exposed, infectious, quarantined, and removed (recovered or dead) compartments, and a set of delay integral equations is used to describe the system dynamics. Time-varying infection rates are allowed in the model to capture the responses to control measures taken, and distributed delay distributions are used to capture variability in individual responses to an infection. The constructed extended compartmental model is a nonlinear dynamical system with distributed delays and time-varying parameters. The critical role of data is elucidated, and it is discussed as to how the compartmental model can be used to capture responses to various measures including quarantining. Data for different parts of the world are considered, and comparisons are also made in terms of the reproductive number. The obtained results can be useful for furthering the understanding of disease dynamics as well as for planning purposes.

Engineering cell-derived extracellular matrix for peripheral nerve regeneration.

Extracellular matrices (ECMs) play a key role in nerve repair and are recognized as the natural source of biomaterials. In parallel to extensively studied tissue-derived ECMs (ts-ECMs), cell-derived ECMs (cd-ECMs) also have the capability to partially recapitulate the complicated regenerative microenvironment of native nerve tissues. Notably, cd-ECMs can avoid the shortcomings of ts-ECMs. Cd-ECMs can be prepared by culturing various cells or even autologous cells in vitro under pathogen-free conditions. And mild decellularization can achieve efficient removal of immunogenic components in cd-ECMs. Moreover, cd-ECMs are more readily customizable to achieve the desired functional properties. These advantages have garnered significant attention for the potential of cd-ECMs in neuroregenerative medicine. As promising biomaterials, cd-ECMs bring new hope for the effective treatment of peripheral nerve injuries. Herein, this review comprehensively examines current knowledge about the functional characteristics of cd-ECMs and their mechanisms of interaction with cells in nerve regeneration, with a particular focus on the preparation, engineering optimization, and scalability of cd-ECMs. The applications of cd-ECMs from distinct cell sources reported in peripheral nerve tissue engineering are highlighted and summarized. Furthermore, current limitations that should be addressed and outlooks related to clinical translation are put forward as well.

Alpaca-derived nanobody targeting the hydrophobic pocket of the HIV-1 gp41 NHR broadly neutralizes HIV-1 by blocking six-helix bundle formation.

The highly conserved hydrophobic pocket region of HIV-1 gp41 NHR triple-stranded coiled coil is crucial for the binding of CHR to NHR to form a six-helix bundle (6-HB). This pocket is only exposed instantaneously during fusion, making it an ideal target for antibody drug design. However, IgG molecule is too big to enter the pocket during the fusion process. Therefore, to overcome the steric hindrance and kinetic obstacles caused by the formation of gp41 pre-hairpin fusion intermediate, we obtained nanobodies (Nbs) targeting NHR by immunizing alpaca with an NHR-trimer mimic. Specifically, we identified a Nb, Nb-172, that exhibited potent and broadly neutralizing activity against HIV-1 pseudoviruses, HIV-1 primary isolates, and T20-resistant HIV-1 strains. In addition, the combinatorial use of mD1.22 and Nb-172 exhibited synergism in inhibiting HIV-1 infection inactivating cell-free virions. Nb-172 can competitively bind to the hydrophobic pocket of gp41 NHR to inhibit 6-HB formation. These findings suggest that Nb-172 merits further investigation as a potential therapeutic for HIV-1 infection.

Accelerated corrosion of 316L stainless steel in a simulated oral environment via extracellular electron transfer and acid metabolites of subgingival microbiota.

316L stainless steel (SS) is widely applied as microimplant anchorage (MIA) due to its excellent mechanical properties. However, the risk that the oral microorganisms can corrode 316L SS is fully neglected. Microbiologically influenced corrosion (MIC) of 316L SS is essential to the health and safety of all patients because the accelerated corrosion caused by the oral microbiota can trigger the release of Cr and Ni ions. This study investigated the corrosion behavior and mechanism of subgingival microbiota on 316L SS by 16S rRNA and metagenome sequencing, electrochemical measurements, and surface characterization techniques. Multispecies biofilms were formed by the oral subgingival microbiota in the simulated oral anaerobic environment on 316L SS surfaces, significantly accelerating the corrosion in the form of pitting. The microbiota samples collected from the subjects differed in biofilm compositions, corrosion behaviors, and mechanisms. The oral subgingival microbiota contributed to the accelerated corrosion of 316L SS via acidic metabolites and extracellular electron transfer. Our findings provide a new insight into the underlying mechanisms of oral microbial corrosion and guide the design of oral microbial corrosion-resistant materials.

Signaling pathways involved in paraquat-induced pulmonary toxicity: Molecular mechanisms and potential therapeutic drugs.

Paraquat poisoning is a severe health problem globally, particularly in developing countries. Due to its severe toxicity, the mortality rate of paraquat poisoning is greatly higher than other pesticide poisoning. Paraquat accumulates in the lung by specific polyamine uptake and causes a great amount of reactive oxygen species generation induced by redox cycling. Free radicals can further cause cellular damage via lipid peroxidation, mitochondrial damage, inflammatory response, and apoptosis in many organs including lung, liver, and kidney. The potential mechanisms of paraquat toxicity in the lung are extremely complicated. In this review, the biochemical mechanisms and pathophysiological process of paraquat-induced pulmonary toxicity are systematically elaborated based on previous studies. Furthermore, the signaling pathways including Nrf2/ARE, NF-κB, NLRP3 inflammasome, TLRs, PPAR-γ, MAPKs, AMPK, Rho/ROCK, PI3K/Akt/mTOR, TGF-ß/Smad, and Wnt/ß-catenin and the potential therapeutic drugs are comprehensively summarized. Further studies are still required to evaluate the efficacy of these drugs in the future.

The Experimental Study of Periodontal Ligament Stem Cells Derived Exosomes with Hydrogel Accelerating Bone Regeneration on Alveolar Bone Defect.

INTRODUCTION: this study was conducted to investigate the osteogenic ability of periodontal ligament stem cells (PDLSCs) derived exosomes (PDLSCs-Exos) and the effect of PDLSCs-Exos with hydrogel on alveolar bone defect repairment in the rat. METHODS: the PDLSCs were obtained through primary cell culture, and PDLSCs-Exos were purified by the ultracentrifugation method. The CCK-8 kit and ALP staining were used to explore the effect of PDLSCs-Exos on promoting the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In vivo, the alveolar bone defect models were made mesial to the bilateral maxillary first molars of rats. MicroCT, HE staining, and Masson staining were used to analyze the new bone at the bone defect of rats. RESULTS: the periodontal ligament stem cells and the periodontal ligament stem cells derived exosomes were successfully extracted. The results of the CCK-8 kit and ALP staining showed PDLSCs-Exos significantly promoted the proliferation osteogenic differentiation of BMSCs. In vivo experiment results revealed that compared with the control group and the hydrogel group, the rats in the hydrogel with exosomes group showed more new bone formation in alveolar bone defects. CONCLUSION: Periodontal ligament stem cells and exosomes derived from periodontal ligament stem cells were successfully extracted. The results demonstrated that the hydrogel successfully delivered periodontal ligament stem cells derived exosomes for repairing alveolar bone defects in rats in vivo at the initial stage.

Inhibition efficiency of 304-Cu stainless steel against oral bacterial biofilm.

PURPOSE: This study aims to evaluate the antibacterial properties of 304 Cu-bearing stainless steel (SS) with different Cu contents (0, 2.5, 4.5 wt.%) against oral biofilms of Streptococcus mutans (S. mutans), Streptococcus sanguinis (S. sanguinis), and their mixture. METHODS: Bacterial biofilms on the surface of 304-Cu SS were characterized by plate counting, 4', 6-diamidino-2-phenylindole (DAPI) staining with aid of sanning electron microscopy (SEM) and 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT). In addition, the inhibition zone method was also employed to evaluate the antibacterial properties of 304-Cu SS. Cell Counting Kit-8 (CCK-8) and flow cytometry were used to assess the cytotoxicity and apoptosis rate of 304-Cu SS, respectively. RESULTS: 304-4.5Cu SS could effectively inhibit the attachment, formation, activity, and metabolism of bacterial biofilm, possessing the best antibacterial properties exceeding 99.9% of antibacterial rate against S. mutans, S. sanguinis, and their mixture. The diameters of inhibition zones to S. mutans and S. sanguinis on the surface of 304-4.5Cu SS were 21.7 and 14.7 mm, respectively. The results of cell experiments in vitro showed that both 304-2.5Cu SS and 304-4.5Cu SS had no evident cytotoxicity with an identical grade 1. The apoptosis rate exhibited a gradually increased tendency with increase of the Cu content in 304 SS. CONCLUSIONS: 304-4.5Cu SS without cytotoxic effect on NIH3T3 cells has obvious antibacterial activity against S. mutans, S. sanguinis and their mixture. CLINICAL SIGNIFICANCE: The Cu-bearing stainless steel provides a new solution to be used as oral orthodontic devices for inhibiting oral microflora imbalance and enamel demineralization.

A Potent Neutralizing Nanobody Targeting the Spike Receptor-Binding Domain of SARS-CoV-2 and the Structural Basis of Its Intimate Binding.

The continuous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) around the world has raised unprecedented challenges to the human society. Antibodies and nanobodies possessing neutralization activity represent promising drug candidates. In this study, we report the identification and characterization of a potent SARS-CoV-2 neutralizing nanobody that targets the viral spike receptor-binding domain (S-RBD). The nanobody, termed as Nb-007, engages SARS-CoV-2 S-RBD with the two-digit picomolar binding affinity and shows outstanding virus entry-inhibition activity. The complex structure of Nb-007 bound to SARS-CoV-2 S-RBD reveals an epitope that is partially overlapping with the binding site for the human receptor of angiotensin-converting enzyme 2 (ACE2). The nanobody therefore exerts neutralization by competing with ACE2 for S-RBD binding, which is further ascertained by our in-vitro biochemical analyses. Finally, we also show that Nb-007 reserves promising, though compromised, neutralization activity against the currently-circulating Delta variant and that fusion of the nanobody with Fc dramatically increases its entry-inhibition capacity. Taken together, these data have paved the way of developing Nb-007 as a drug-reserve for potential treatment of SARS-CoV-2 related diseases.

Discovery and synthesis of novel luteolin derivatives as DAT agonists.

Luteolin, 5,7-dihydroxy-2-(3,4-dihydroxyphenyl)-4H-chromen-4-one, has been proposed and proved to be a novel dopamine transporter (DAT) activator. In order to develop this potential of luteolin, a series of novel luteolin derivatives were designed, synthesized, and evaluated for their DAT agonistic activities, utilizing constructed Chinese hamster ovary (CHO) cell lines stably expressing rat DAT. Biological screening results demonstrated that luteolin derivatives 1d, 1e, and 4c carry great DAT agonistic potency (EC(50)=0.046, 0.869, and 1.375µM, respectively) compared with luteolin 8 (EC(50)=1.45±0.29µM). Luteolin derivative 1d, notably, exhibited a 32-fold-higher DAT agonistic potency than luteolin. These luteolin derivatives represent a novel DAT agonist class, from which lead compounds useful for exploration of additional novel DAT agonists could be drawn.

Association Between Consecutive Ambient Air Pollution and Chronic Obstructive Pulmonary Disease Hospitalization: Time Series Study During 2015-2017 in Chengdu China.

This paper investigates the association between consecutive ambient air pollution and Chronic Obstructive Pulmonary Disease (COPD) hospitalization in Chengdu China. The three-year (2015-2017) time series data for both ambient air pollutant concentrations and COPD hospitalizations in Chengdu are approved for the study. The big data statistic analysis shows that Air Quality Index (AQI) exceeded the lighted air polluted level in Chengdu region are mainly attributed to particulate matters (i.e., PM2.5 and PM10). The time series study for consecutive ambient air pollutant concentrations reveal that AQI, PM2.5, and PM10 are significantly positive correlated, especially when the number of consecutive polluted days is greater than nine days. The daily COPD hospitalizations for every 10 µg/m3 increase in PM2.5 and PM10 indicate that consecutive ambient air pollution can lead to an appearance of an elevation of COPD admissions, and also present that dynamic responses before and after the peak admission are different. Support Vector Regression (SVR) is then used to describe the dynamics of COPD hospitalizations to consecutive ambient air pollution. These findings will be further developed for region specific, hospital early notifications of COPD in responses to consecutive ambient air pollution.

Polycomb Protein OsFIE2 Affects Plant Height and Grain Yield in Rice.

Polycomb group (PcG) proteins have been shown to affect growth and development in plants. To further elucidate their role in these processes in rice, we isolated and characterized a rice mutant which exhibits dwarfism, reduced seed setting rate, defective floral organ, and small grains. Map-based cloning revealed that abnormal phenotypes were attributed to a mutation of the Fertilization Independent Endosperm 2 (OsFIE2) protein, which belongs to the PcG protein family. So we named the mutant as osfie2-1. Histological analysis revealed that the number of longitudinal cells in the internodes decreased in osfie2-1, and that lateral cell layer of the internodes was markedly thinner than wild-type. In addition, compared to wild-type, the number of large and small vascular bundles decreased in osfie2-1, as well as cell number and cell size in spikelet hulls. OsFIE2 is expressed in most tissues and the coded protein localizes in both nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that OsFIE2 interacts with OsiEZ1 which encodes an enhancer of zeste protein previously identified as a histone methylation enzyme. RNA sequencing-based transcriptome profiling and qRT-PCR analysis revealed that some homeotic genes and genes involved in endosperm starch synthesis, cell division/expansion and hormone synthesis and signaling are differentially expressed between osfie2-1 and wild-type. In addition, the contents of IAA, GA3, ABA, JA and SA in osfie2-1 are significantly different from those in wild-type. Taken together, these results indicate that OsFIE2 plays an important role in the regulation of plant height and grain yield in rice.

Chemical characteristics of saponins from Paris fargesii var. brevipetala and cytotoxic activity of its main ingredient, paris saponin H.

More attention was paid to the anti-tumor activity of Rhizoma Paridis (RP) recently, of which the wild resource was decreased significantly. This study was aimed to elucidate the chemical characteristics of Paris fargesii var. brevipetala (PFB) that may be administrated as alternate resource of legal RP. A HPLC-ELSD method was established to characterize the steroid saponins in rhizomes of PFB and two legal Paris species [Paris polyphylla var. chinensis (PPC) and P. polyphylla var. yunnanensis (PPY)] in Chinese Pharmacopoeia (CP). Ten saponins (paris saponins I, II, V, VI, VII, H, gracillin and other three paris saponins) were involved as standards. The results indicated that PFB contained pennogenyl saponins as the main components with small amounts of diosgenin saponins. The total contents of the detected saponins in PFB ranged from 9.12mg/g to 85.33mg/g. Nine of the twelve PFB samples own a total content of paris saponins I, II, VI, and VII more than 6.0mg/g (meeting the standard of CP 2010 edition). Principal Component Analysis (PCA) and Partial Least Squares-Discriminate Analysis (PLS-DA) both confirmed the fact that saponin profiles of PFB, PPC and PPY were different from each other. In addition, paris saponin H (Ps H), the predominant saponin of PFB (>50%), was tested in vitro to evaluate its cytotoxic activities on HepG2, A549, RPE and L929 cells with a positive control of Cisplatin. Ps H showed a remarkable cytotoxic activity on A549 cells with an IC(50) value of 1.53±0.08µg/mL.

Application of microscopy in authentication and distinguishing of 11 Paris species in West Sichuan.

In the trade and raw drug market, the medicinal plants of genus Paris are available in the form of rhizomes without any vouchers, thus making it difficult to identify and distinguish the different species of Genus Paris. Recent studies have shown that the species of Paris possess different chemical constituents, pharmacological activities, and efficiencies in clinical application. To distinguish 11 species of Paris collected from the western Sichuan province of China and ensure their safety and efficacy, in the present work, the microscopic characteristics of rhizomes and the crude drug powder of the 11 species of Paris were compared using a light microscope according to the usual microscopic techniques. The results of the microscopic features were systematically described and illustrated. The differences among these species are great enough that the identity of most material can be easily determined. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters based on these anatomic characteristics analyzed was drawn up and presented for the Paris species studied. The study indicated that light microscopy and related techniques could provide a method that is convenient, feasible, and can be unambiguously applied in the authentication of species of Paris. This information is required not only for the identification procedures that guarantee the utilization of the appropriate raw material, but also for the quality control standards demanded.

Synthesis and biological evaluation of (R)-N-(diarylmethylthio/sulfinyl)ethyl/propyl-piperidine-3-carboxylic acid hydrochlorides as novel GABA uptake inhibitors.

A series of new (R)-1-(2-diarylmethylthio/sulfinyl)ethyl-piperidine-3-carboxylic acid hydrochlorides 5a-d/6a-d and (R)-1-(3-diarylmethylthio)propyl-piperidine-3-carboxylic acid hydrochlorides 5'a-d were synthesized and evaluated as gamma-aminobutyric acid uptake inhibitors through cultured cell lines expressing mouse GAT1. Biological screening results demonstrated that the compounds 6a-d with diarylmethylsulfinyl ethyl side chain show more potent GAT1 inhibitory activities than 5a-d/5'a-d with diarylmethylthio ethyl/propyl moieties. Some of them, such as 6a, exhibited excellent inhibitions of [(3)H]-GABA uptake in cultured cells, which is 496-fold higher than (R)-nipecotic acid and 11.5 times less than tiagabine. The synthesis and structure-activity relationships are discussed.