Sensitive Dual-Signal ELISA Based on Specific Phage-Displayed Double Peptide Probes with Internal Filtering Effect to Assay Monkeypox Virus Antigen.

The global spread of monkeypox has become a worldwide public healthcare issue. Therefore, there is an urgent need for accurate and sensitive detection methods to effectively control its spreading. Herein, we screened by phage display two peptides M4 (sequence: DPCGERICSIAL) and M6 (sequence: SCSSFLCSLKVG) with good affinity and specificity to monkeypox virus (MPXV) B21R protein. To simulate the state of the peptide in the phage and to avoid spatial obstacles of the peptide, GGGSK was added at the C terminus of M4 and named as M4a. Molecular docking shows that peptide M4a and peptide M6 are bound to different epitopes of B21R by hydrogen bonds and salt-bridge interactions, respectively. Then, peptide M4a was selected as the capture probe, phage M6 as the detection probe, and carbonized polymer dots (CPDs) as the fluorescent probe, and a colorimetric and fluorescent double-signal capture peptide/antigen/signal peptide-displayed phage sandwich ELISA triggered by horseradish peroxidase (HRP) through a simple internal filtration effect (IFE) was constructed. HRP catalyzes H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB, which can further quench the fluorescence of CPDs through IFE, enabling to detect MPXV B21R in colorimetric and fluorescent modes. The proposed simple immunoassay platform shows good sensitivity and reliability in MPXV B21R detection. The limit of detection for colorimetric and fluorescent modes was 27.8 and 9.14 pg/mL MPXV B21R, respectively. Thus, the established double-peptide sandwich-based dual-signal immunoassay provides guidance for the development of reliable and sensitive antigen detection capable of mutual confirmation, which also has great potential for exploring various analytical strategies for other respiratory virus surveillance.

Tradeoff between productivity and stability across above- and below-ground communities.

An 11-year nitrogen addition experiment reveals that for both plants and soil microorganisms, the ruderal strategists had higher productivity but lower stability, while the tolerant strategists had higher stability and lower productivity, leading to the tradeoff between productivity and stability within and across above- and below-ground communities.

An ultrasensitive ELISA to assay femtomolar level SARS-CoV-2 antigen based on specific peptide and tyramine signal amplification.

The SARS-CoV-2 spreading rapidly has aroused catastrophic public healthcare issues and economy crisis worldwide. It plays predominant role to rapidly and accurately diagnose the virus for effective prevention and treatment. As an abundant transmembrane protein, spike protein (SP) is one of the most valuable antigenic biomarkers for diagnosis of COVID-19. Herein a phage expression of WNLDLSQWLPPM peptide specific to SARS-CoV-2 SP was screened. Molecular docking revealed that the isolated peptide binds to major antigenic epitope locating at S2 subunit with hydrogen bonding. Taking the specific peptide as antigen sensing probe and tyramine signal amplification (TSA), an ultrasensitive "peptide-antigen-antibody" ELISA (p-ELISA) was explored, by which the limit of detection (LOD) was 14 fM and 2.8 fM SARS-CoV-2 SP antigen for first TSA and secondary TSA, respectively. Compared with the LOD by the p-ELISA by direct mode, the sensitivity with 2nd TSA enhanced 100 times. Further, the proposed p-ELISA method can detect SARS-CoV-2 pseudoviruses down to 10 and 3 TCID50/mL spiked in healthy nasal swab sample with 1st TSA and 2nd TSA, separately. Thus, the proposed p-ELISA method with TSA is expected to be a promising ultrasensitive tool for rapidly detecting SARS-CoV-2 antigen to help control the infectious disease.

Discovery of a Phage Peptide Specifically Binding to the SARS-CoV-2 Spike S1 Protein for the Sensitive Phage-Based Enzyme-Linked Chemiluminescence Immunoassay of the SARS-CoV-2 Antigen.

The COVID-19 pandemic has led to a global crisis with devastating effects on public healthcare and the economy. Sensitive detection of SARS-CoV-2 is the key to diagnose and control its spread. The spike (S) protein is an abundant viral transmembrane protein and a suitable target protein for the selective recognition of SARS-CoV-2. Here, we report that with bovine serum albumin prescreening, a specific phage peptide targeting SARS-CoV-2 S1 protein was biopanned with the pIII phage display library. The identified phage #2 expressing the peptide (amino acid sequence: NFWISPKLAFAL) shows high affinity to the target with a dissociation constant of 3.45 ± 0.58 nM. Furthermore, the identified peptide shows good specificity with a binding site at the N-terminal domain of the S1 subunit through a hydrogen bond network and hydrophobic interaction, supported by molecular docking. Then, a sandwiched phage-based enzyme-linked chemiluminescence immunoassay (ELCLIA) was established by using phage #2 as a bifunctional probe capable of SARS-CoV-2 S1 antigen recognition and signal amplification. After optimizing the conditions, the proposed phage ELCLIA exhibited good sensitivity, and as low as 78 pg/mL SARS-CoV-2 S1 could be detected. This method can be applied to detect as low as 60 transducing units (TU)/mL SARS-CoV-2 pseudovirus in 50% saliva. Therefore, specific phage peptides have good prospects as powerful biological recognition probes for immunoassay detection and biomedical applications.

Variations of tongue coating microbiota in children with Henoch-Schönlein purpura nephritis.

BACKGROUND: Variations in the oral microbiota have been significantly correlated with the progress of autoimmune diseases, such as immunoglobulin A nephropathy and Henoch-Schönlein purpura (HSP). However, there is no report outlining the character of tongue coating microbiota variations in children with Henoch-Schönlein purpura nephritis (HSPN). METHOD: A total of 20 children with HSPN and 14 healthy controls were recruited for this research. Tongue coating samples of two groups were collected for 16S rRNA gene sequencing. The diversity, principal component analysis (PCA), nonmetric multidimensional scaling (NMDS), partial least squares discriminant analysis (PLS-DA), and linear discriminant analysis (LDA) effect size (LEfSe) were performed. Microbial function was assessed using the PICRUST. RESULTS: The ACE and Chao indices were greatly lower in the HSPN group than in the HG (P = 0.001). The Shannon and Simpson indices were dramatically reduced in children with HSPN compared with those in the healthy controls (P = 0.005). Bacteroidales, Selenomonadales, Lactobacillales, Fusobacteriales, Neisseriales, and Actinomycetales composed more than 80% of all sequences, while Bacteroidales was the most generous order in both groups. PCA, NMDS and PLS-DA showed a marked difference between the control and HSPN groups. LEfSe analysis showed alteration of tongue coating microbiota in the HSPN group. There were 30 metabolic functions significantly differed between the two groups. CONCLUSIONS: Children with HSPN have substantially various tongue coating microbiota compared to healthy controls. Even though this research does not indicate causality, it is beneficial to enhance the possibility for coming microbial-based treatments to enhance the clinical effects of HSPN in children.

Nitrogen deposition mediates more stochastic processes in structuring plant community than soil microbial community in the Eurasian steppe.

Anthropogenic environmental changes may affect community assembly through mediating both deterministic (e.g., competitive exclusion and environmental filtering) and stochastic processes (e.g., birth/death and dispersal/colonization). It is traditionally thought that environmental changes have a larger mediation effect on stochastic processes in structuring soil microbial community than aboveground plant community; however, this hypothesis remains largely untested. Here we report an unexpected pattern that nitrogen (N) deposition has a larger mediation effect on stochastic processes in structuring plant community than soil microbial community (those <2 mm in diameter, including archaea, bacteria, fungi, and protists) in the Eurasian steppe. We performed a ten-year nitrogen deposition experiment in a semiarid grassland ecosystem in Inner Mongolia, manipulating nine rates (0-50 g N m-2 per year) at two frequencies (nitrogen added twice or 12 times per year) under two grassland management strategies (fencing or mowing). We separated the compositional variation of plant and soil microbial communities caused by each treatment into the deterministic and stochastic components with a recently-developed method. As nitrogen addition rate increased, the relative importance of stochastic component of plant community first increased and then decreased, while that of soil microbial community first decreased and then increased. On the whole, the relative importance of stochastic component was significantly larger in plant community (0.552±0.035; mean±standard error) than in microbial community (0.427±0.035). Consistently, the proportion of compositional variation explained by the deterministic soil and community indices was smaller for plant community (0.172-0.186) than microbial community (0.240-0.767). Meanwhile, as nitrogen addition rate increased, the linkage between plant and microbial community composition first became weaker and then became stronger. The larger stochasticity in plant community relative to microbial community assembly suggested that more stochastic strategies (e.g., seeds addition) should be adopted to maintain above- than below-ground biodiversity under the pressure of nitrogen deposition.

Core bacteria and fungi in response to residue retention and their contribution to soil multifunctionality in maize agroecosystem.

Core microbiome has been proven to play crucial roles in soil function. However, we still lack knowledge on how core microbiome responds to crop residue retention, and whether they contribute to this process. Consequently, we examined the effect of residue retention on soil core and non-core microbial communities in maize seedling, mature stage and freezing period based on a multi-site field experiment in Sanjiang Plain, Northeast China. Totally, 247 bacterial amplicon sequence variants (ASVs) and 109 fungal ASVs were identified as core microbiota. Both core and non-core bacterial/fungal community composition were significantly influenced by residue retention across all study sites. Especially, the core fungal community shifted towards a saprotroph-dominated community. Normalized stochastic ratio pattern revealed that that deterministic process dominated both core and non-core microbial community assembly processes. Residue retention enhanced the deterministic process of core microbial community assembly, while exhibited opposite effect on non-core microbial community. This study also revealed that soil fungi were more sensitive to residue retention than bacteria, with a larger proportion of core fungi were enriched or depleted by residue retention. In addition, residue retention complicated core bacterial co-occurrence network, while simplified fungal network. Our results pointed out both no reduction in microbial diversity or collapse in microbial network structure after repeated freezing-thawing cycles. The potential function of core microbiome was evaluated through random forest analysis and structural equation model, the results indicated core microbiome contributed more to multifunctionality than non-core microbiome. Overall, this study strengthened our understanding of soil core microbiome in response to residue retention, and highlighted their importance in maintaining soil multifunctionality.

Diagnostics and analysis of SARS-CoV-2: current status, recent advances, challenges and perspectives.

The disastrous spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has induced severe public healthcare issues and weakened the global economy significantly. Although SARS-CoV-2 infection is not as fatal as the initial outbreak, many infected victims suffer from long COVID. Therefore, rapid and large-scale testing is critical in managing patients and alleviating its transmission. Herein, we review the recent advances in techniques to detect SARS-CoV-2. The sensing principles are detailed together with their application domains and analytical performances. In addition, the advantages and limits of each method are discussed and analyzed. Besides molecular diagnostics and antigen and antibody tests, we also review neutralizing antibodies and emerging SARS-CoV-2 variants. Further, the characteristics of the mutational locations in the different variants with epidemiological features are summarized. Finally, the challenges and possible strategies are prospected to develop new assays to meet different diagnostic needs. Thus, this comprehensive and systematic review of SARS-CoV-2 detection technologies may provide insightful guidance and direction for developing tools for the diagnosis and analysis of SARS-CoV-2 to support public healthcare and effective long-term pandemic management and control.

Biopanning of specific peptide for SARS-CoV-2 nucleocapsid protein and enzyme-linked immunosorbent assay-based antigen assay.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide which triggered serious public health issues. The search for rapid and accurate diagnosis, effective prevention, and treatment is urgent. The nucleocapsid protein (NP) of SARS-CoV-2 is one of the main structural proteins expressed and most abundant in the virus, and is considered a diagnostic marker for the accurate and sensitive detection of SARS-CoV-2. Herein, we report the screening of specific peptides from the pIII phage library that bind to SARS-CoV-2 NP. The phage monoclone expressing cyclic peptide N1 (peptide sequence, ACGTKPTKFC, with C&C bridged by disulfide bonding) specifically recognizes SARS-CoV-2 NP. Molecular docking studies reveal that the identified peptide is bound to the "pocket" region on the SARS-CoV-2 NP N-terminal domain mainly by forming a hydrogen bonding network and through hydrophobic interaction. Peptide N1 with the C-terminal linker was synthesized as the capture probe for SARS-CoV-2 NP in ELISA. The peptide-based ELISA was capable of assaying SARS-CoV-2 NP at concentrations as low as 61 pg/mL (∼1.2 pM). Furthermore, the as-proposed method could detect the SARS-CoV-2 virus at limits as low as 50 TCID50 (median tissue culture infective dose)/mL. This study demonstrates that selected peptides are powerful biomolecular tools for SARS-CoV-2 detection, providing a new and inexpensive method of rapidly screening infections as well as rapidly diagnosing coronavirus disease 2019 patients.

Disturbance-level-dependent post-disturbance succession in a Eurasian steppe.

Anthropogenic disturbances may decrease as we take measures to control them. However, the patterns and mechanisms of post-disturbance ecosystem succession have rarely been studied. Here we reported that disturbance level determined the importance of stochastic relative to deterministic changes in ecosystem components (plant community composition, soil microbial community composition, and soil physicochemical indices), and thus predefined the pattern of post-disturbance ecosystem succession. We proposed a theoretical framework with five disturbance levels corresponding to distinct succession patterns. We conducted a nitrogen addition experiment in a temperate steppe, monitored these ecosystem components during "disturbance" treatment (2010-2014) and post-treatment "succession" (2014-2018). The disturbance level experienced by each component in each treatment was inferred by fitting the observed succession patterns into the theoretical framework. The mean disturbance level of these components was found to increase quadratically with nitrogen addition rate. This was because increasing nitrogen addition reduced the importance of stochastic relative to deterministic changes in these components, and these changes had a quadratic relationship with disturbance level. Overall, our results suggested that by monitoring the importance of stochastic relative to deterministic changes in an ecosystem, we can estimate disturbance levels and predict succession patterns, as well as propose disturbance-level-dependent strategies for post-disturbance restoration.

Distinctive pattern and mechanism of precipitation changes affecting soil microbial assemblages in the Eurasian steppe.

Precipitation may increase or decrease by different intensities, but the pattern and mechanism of soil microbial community assembly under various precipitation changes remain relatively underexplored. Here, although ±30% precipitation caused a small decrease (∼19%) in the within-treatment taxonomic compositional dissimilarity through the deterministic competitive exclusion process in a steppe ecosystem, ±60% precipitation caused a large increase (∼35%) in the dissimilarity through the stochastic ecological drift process (random birth/death), which was in contrast with the traditional thought that increasing the magnitude of environmental changes (e.g., from +30% to +60%) would elevate the importance of deterministic relative to stochastic processes. The increased taxonomic dissimilarity/stochasticity under ±60% precipitation translated into functional dissimilarity/stochasticity at the gene, protein, and enzyme levels. Overall, our results revealed the distinctive pattern and mechanism of precipitation changes affecting soil microbial community assembly and demonstrated the need to integrate microbial taxonomic information to better predict their functional responses to precipitation changes.

Differential expression of long non-coding RNA and mRNA in children with Henoch-Schönlein purpura nephritis.

Long non-coding RNAs (lncRNAs) serve an essential role in regulating immunological functions. However, their impact on Henoch-Schönlein purpura nephritis (HSPN), has remained elusive. The present study determined the expression of lncRNAs and mRNAs in the peripheral blood of 6 children with HSPN and recruited 4 healthy children for comparative study. High-throughput sequencing revealed outstanding differences in lncRNA and mRNA expression, which were verified through reverse transcription-quantitative polymerase chain reaction. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to investigate the associated biological functions and possible mechanisms of lncRNAs and mRNAs in HSPN. A total of 820 differentially expressed lncRNAs between the two groups were identified, of which 34 were upregulated and 786 were downregulated. Simultaneously, a total of 3,557 mRNAs were also identified to be differentially expressed, of which 1,232 were upregulated and 2,325 were downregulated. The results revealed that the expression of lncRNAs including ENST00000378432, ENST00000571370, uc001kfc.1 and uc010qna.2 was decreased in HSPN patients compared with that in healthy controls. These lncRNAs were associated with the p53 signaling pathway and apoptosis-associated genes (AKT2, tumor protein 53, phosphatase and tensin homolog and FAS). Further studies of those lncRNAs will be performed to elucidate their functions in apoptosis. Complete raw data files were deposited in the Gene Expression Omnibus (GEO) at National Center for Biotechnology information under the GEO accession no. GSE102114 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE102114).

Chinese herbal medicine for the treatment of Henoch-Schönlein purpura nephritis in children: A prospective cohort study protocol.

INTRODUCTION: Henoch-Schönlein purpura nephritis (HSPN) involves the renal impairment of Henoch-Schönlein purpura and can easily relapse into life-threatening late nephropathy in severe cases. Although there is a lack of validated evidence for its effectiveness, Chinese herbal medicine (CHM) is one of the most commonly used methods in China to treat HSPN. It is thus need to report the protocol of a prospective cohort trial using CHM to investigate the effectiveness, safety and advantages for children with HSPN. METHODS AND ANALYSIS: This large, prospective, multicenter cohort study started in May 2015 in Shenyang. Six hundred children diagnosed with HSPN were recruited from 3 institutions and are followed-up every 2 to 4 weeks till May 2020. Detailed information of participants includes general information, history of treatment, physical examination, and symptoms of TCM is taken face-to-face at baseline. ETHICS AND DISSEMINATION: This study has received ethical approval from the ethics committee of institutional review board of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine (No.2016CS(KT)-002-01). Articles summarizing the primary results and ancillary analyses will be published in peer-reviewed journals. TRIAL REGISTRATION: Clinical Trials Registration: NCT02878018.

Reconstruction of full-thickness buccal defects with folded radial forearm flaps: A retrospective clinical study.

Our goal was to describe our experience of the folded radial forearm flap (RFF) flap in through-and-through buccal defect reconstructions.Patients who had received a folded RFF flap for full-thickness cheek defect reconstruction were included. The flap success rate and functional results were evaluated.Six patients were enrolled. All flaps survived totally without any complication; the mean flap size was 75.5 (range 32-135) cm. The mean mouth-open width was 4.2 (range 3.5-4.7) cm at 6 months after operation. All patients were satisfied with the appearance and were capable of maintaining a regular oral diet, and no patients complained of an inability to eat in a public setting, microstomia, or drooling.Radial forearm flap was a reliable method for through-and-through buccal reconstruction with high success rate and good functional result.

Management of and risk factors for regional recurrence in upper lip squamous cell carcinoma.

Our goal was to evaluate the risk factors for regional recurrence in upper lip squamous cell carcinoma (SCC).A total of 109 patients with upper lip SCC were retrospectively enrolled. Clinical and pathologic information was collected and analyzed.Fifteen (13.8%) patients had a regional metastasis, 2 patients had parotid node metastasis, and no local or distant metastasis was noted. There was neck level I node metastasis in 15 (100%) patients. In multivariate analysis, invasion depth and degree of differentiation were significantly associated with regional metastasis. The 5- and 10-year disease-specific survival rate was 95.4% and 80.1%, respectively.Invasion depth and degree of differentiation have reliable value for predicting regional metastasis.

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Relying on a long term field manipulated grazing experiment of typical grassland in Inner Mongolia, China, we investigated the responses of soil organic carbon, total nitrogen, extractable carbon and nitrogen, and microbial biomass carbon and nitrogen to livestock grazing and grass mo-wing. The results showed that grazing decreased extractable organic carbon by 11.4%-37.1%, while mowing increased extractable organic carbon by 5.8%. Grazing and mowing increased extrac table nitrogen by 10%-340% and 10%-240%, respectively. Grazing intensity less than 6.0 sheep·hm-2 favored the maintenance of microbial biomass carbon, and heavy grazing intensity, i.e. , 7.5 and 9.0 sheep·hm-2, decreased microbial biomass carbon. Mowing consistently increased microbial biomass carbon and nitrogen by 31.0% and 9.8%, respectively. Path analysis showed that soil organic carbon, extractable total nitrogen and total nitrogen were the dominant controls of microbial biomass carbon with soil organic carbon and total extractable nitrogen as the direct influencing factors. Soil pH, extractable organic carbon, soil organic carbon and total extractable nitrogen were the dominant controls of microbial biomass nitrogen with soil organic carbon and total extractable nitrogen as the direct factors. Mowing, light grazing and moderate grazing were beneficial to maintaining or improving soil function, and heavy grazing would cause soil degradation.

Correction: Synergistic and targeted drug delivery based on nano-CeO2 capped with galactose functionalized pillar[5]arene via host-guest interactions.

Correction for 'Synergistic and targeted drug delivery based on nano-CeO2 capped with galactose functionalized pillar[5]arene via host-guest interactions' by Xiaowen Wu et al., J. Mater. Chem. B, 2017, 5, 3483-3487.

Synergistic and targeted drug delivery based on nano-CeO2 capped with galactose functionalized pillar[5]arene via host-guest interactions.

A smart drug delivery system based on porous CeO2 nano-rods (CeONRs) capped with galactose functionalized pillar[5]arene via host-guest interactions has been constructed, which showed GSH-responsiveness, synergism with anticancer drugs and cancer targeting ability resulting from its disulphide unit, ceria properties and galactose units, respectively.