Microbial Communities and Functional Genes in Periodontitis and Healthy Controls.

BACKGROUND: Periodontitis is a chronic progressive disease and the leading cause of tooth loss in adults. Recent studies have shown the impact of oral microbial communities on systemic health and diseases such as cancer, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, diabetes, hypertension, and Alzheimer's disease. In previous case control studies investigatin the relationship between periodontal disease and the oral microbiota, little attention has been paid to the intersections of these domains. METHODS: Here, we used high-throughput 16S rRNA sequencing to analyse the differences in the microbial composition in saliva between a group of patients with chronic periodontitis (C; n = 51) and a healthy control group (H; n = 61) and predicted the functional gene composition by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. RESULTS: We found significant alterations in oral microbial diversity between C and H (P = 0.002). Sixteen genera were significantly different between C and H, and 15 of them were enriched in C linear discriminant analysis (LDA > 2). Fifty functional genes were significantly different between C and H, and 34 of them were enriched in C (P < .025). CONCLUSIONS: Periodontitis is associated with significant changes in the oral microbial community.

Identification of a Linear B Cell Epitope on p54 of African Swine Fever Virus Using Nanobodies as a Novel Tool.

African swine fever (ASF) has received great attention from the swine industry due to the pandemic and the lack of vaccines or effective treatments. In the present study, 13 African swine fever virus (ASFV) p54-specific nanobodies (Nbs) were successfully screened based on Bactrian camel immunization of p54 protein and phage display technology, and their reactivity with the p54 C-terminal domain (p54-CTD) was determined; however, only Nb8-horseradish peroxidase (Nb8-HRP) exhibited the best reactivity. Immunoperoxidase monolayer assay (IPMA) and immunofluorescence assay (IFA) results indicated that Nb8-HRP specifically reacted with ASFV-infected cells. Then, the possible epitopes of p54 were identified using Nb8-HRP. The results showed that Nb8-HRP could recognize p54-CTD truncated mutant p54-T1. Then, 6 overlapping peptides covering p54-T1 were synthesized to determine the possible epitopes. Dot blot and peptide-based enzyme-linked immunosorbent assay (ELISA) results suggested that one novel minimal linear B cell epitope, 76QQWVEV81, which had never been reported before, was identified. Alanine-scanning mutagenesis revealed that 76QQWV79 was the core binding site for Nb8. Epitope 76QQWVEV81 was highly conserved among genotype II ASFV strains and could react with inactivated ASFV antibody-positive serum from naturally infected pigs, indicating that it was a natural linear B cell epitope. These findings provide valuable insights for vaccine design and p54 as an effective diagnostic tool. IMPORTANCE The ASFV p54 protein plays an important role in inducing neutralization antibodies in vivo after viral infection and is often used as a candidate protein for subunit vaccine development. The full understanding of the p54 protein epitope provides a sufficient theoretical basis for p54 as a vaccine candidate protein. The present study uses a p54-specific nanobody as a probe to identify a highly conserved antigenic epitope, 76QQWVEV81, among different ASFV strains, and it can induce humoral immune responses in pigs. This is the first report using virus-specific nanobodies as a tool to identify some special epitopes that cannot be recognized by conventional monoclonal antibodies. This study opens up nanobodies as a new tool for identifying epitopes and also provides a theoretical basis for understanding p54-induced neutralizing antibodies.

Physicochemical and pozzolanic properties of municipal solid waste incineration fly ash with different pretreatments.

Swelling caused by gas generated from municipal solid waste incineration fly ash (MSWIFA) when it is mixed with alkali limits its uses. Besides, the leaching of anion salts and heavy metals contained in MSWIFA poses a high risk to environment. This study presents the feasibility of a one-step alkaline washing, one-step thermal quenching and two-step combination of alkaline washing and thermal quenching pretreatment methods in altering the key properties of MSWIFA for promoting its reusability. It was found that apart from H2(gas), NH3(gas) was also generated during the alkaline washing of the MSWIFA. Besides, pretreatments led to the reduction in particle size, the increase in pore volume and specific surface area of the MSWIFA, as well as the removal of chloride and sulfate anions. All the pretreatment methods were effective in reducing leaching of heavy metals to below levels of nonhazardous waste except Cd and Pb with alkaline washing. Furthermore, both the chemical Frattini test and the mechanical activity index test showed improvement in pozzolanic activities of the MSWIFA after the pretreatments. Overall, the combined pretreatment method was most effective in eliminating gas emission, and reducing leaching of metal ions and anions from the ash, while enhancing the pozzolanic activity of the ash.

TAT Nanobody Exerts Antiviral Effect against PRRSV In Vitro by Targeting Viral Nucleocapsid Protein.

Porcine reproductive and respiratory syndrome (PRRS) is caused by the PRRS virus (PRRSV), which has brought huge economic losses to the pork industry worldwide since its first discovery in the late 1980s in North America. To date, there are no effective commercial vaccines or therapeutic drugs available for controlling the spread of PRRSV. Due to their unique advantages of high affinity and high specificity, nanobodies (Nbs) have received increasing attention in the process of disease diagnosis and treatment. Trans-activator transcription (TAT) can serve as a vector to carry specific proteins into cells by passing through cell membranes. In our previous study, a specific Nb against the PRRSV nucleocapsid (N) protein was screened using phage display technology. For this study, we developed a novel recombinant protein constituting a TAT-conjugated Nb, which we call TAT-Nb1. The target cell entry efficiency of TAT-Nb1 and its effect on PRRSV infection and replication were then investigated. Our results indicate that TAT delivered Nb1 into Marc-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner. Furthermore, TAT-Nb1 dose-dependently suppressed PRRSV infection and replication, where this antiviral effect was independent of PRRSV strain. Co-immunoprecipitation results revealed that Nb1 efficiently interacted with the N protein of PRRSV. Taken together, the presented results suggest that TAT-Nb1 can effectively suppress PRRSV replication, and it may be considered as a new anti-PRRSV candidate drug.

K205R specific nanobody-horseradish peroxidase fusions as reagents of competitive ELISA to detect African swine fever virus serum antibodies.

BACKGROUND: African swine fever virus (ASFV) is a highly contagious hemorrhagic disease and often lethal, which has significant economic consequences for the swine industry. Due to lacking of commercial vaccine, the prevention and control of ASF largely depend on early large-scale detection and screening. So far, the commercial ELISA kits have a long operation time and are expensive, making it difficult to achieve large-scale clinical applications. Nanobodies are single-domain antibodies produced by camelid animals, and have unique advantages such as smaller molecular weight, easy genetic engineering modification and low-costing of mass production, thus exhibiting good application prospects. RESULTS: The present study developed a new method for detection of ASFV specific antibodies using nanobody-horseradish peroxidase (Nb-HRP) fusion proteins as probe. By using camel immunization, phage library construction and phage display technology, five nanobodies against K205R protein were screened. Then, Nb-HRP fusion proteins were produced using genetic modification technology. Based on the Nb-HRP fusion protein as specific antibodies against K205R protein, a new type of cELISA was established to detect ASFV antibodies in pig serum. The cut-off value of the cELISA was 34.8%, and its sensitivity, specificity, and reproducibility were good. Furthermore, the developed cELISA exhibited 99.3% agreement rate with the commercial available ELISA kit (kappa value = 0.98). CONCLUSIONS: The developed cELISA method has the advantages of simple operation, rapid and low-costing, and can be used for monitoring of ASFV infection in pigs, thus providing a new method for the prevention and control of ASF.

Porcine reproductive and respiratory syndrome virus non-structural protein 4 cleaves guanylate-binding protein 1 via its cysteine proteinase activity to antagonize GBP1 antiviral effect.

Porcine reproductive and respiratory syndrome (PRRS) is a highly infectious disease caused by PRRS virus (PRRSV) that causes great economic losses to the swine industry worldwide. PRRSV has been recognized to modulate the host antiviral interferon (IFN) response and downstream interferon-stimulated gene expression to intercept the antiviral effect of host cells. Guanylate-binding proteins (GBPs) are IFN-inducible GTPases that exert broad antiviral activity against several DNA and RNA viruses, of which GBP1 is considered to play a pivotal role. However, the role of GBP1 in PRRSV replication remains unknown. The present study showed that overexpression of GBP1 notably inhibited PRRSV infection, while the knockdown of endogenous GBP1 promoted PRRSV infection. The K51 and R48 residues of GBP1 were essential for the suppression of PRRSV replication. Furthermore, GBP1 abrogated PRRSV replication by disrupting normal fibrous actin structures, which was indispensable for effective PRRSV replication. By using a co-immunoprecipitation assay, we found that GBP1 interacted with the non-structural protein 4 (nsp4) protein of PRRSV, and this interaction was mapped to the N-terminal globular GTPase domain of GBP1 and amino acids 1-69 of nsp4. PRRSV infection significantly downregulated GBP1 protein expression in Marc-145 cells, and nsp4, a 3C-like serine proteinase, was responsible for GBP1 cleavage, and the cleaved site was located at glutamic acid 338 of GBP1. Additionally, the anti-PRRSV activity of GBP1 was antagonized by nsp4. Taken together, these findings expand our understanding of the sophisticated interaction between PRRSV and host cells, PRRSV pathogenesis and its mechanisms of evading the host immune response.

Case Report: Metagenomics Next-Generation Sequencing Can Help Define the Best Therapeutic Strategy for Brain Abscesses Caused by Oral Pathogens.

Brain abscesses are associated with an increased long-term risk of new seizures and increased mortality within several years after infection. Common microorganisms that cause brain abscesses include bacteria, fungi, and mycoplasma. We report a 75-year-old man with a brain abscess caused by Prevotella denticola, an oral pathogen. Based on the clinical condition, we suspected that the patient had a blood-borne brain abscess, and he received antibiotics and systemic supportive treatment. The patient developed shock for the second time after negative Gram-staining results. Metagenomics next-generation sequencing showed one strain from the oral microbiome, confirming our hypothesis, and targeted antibiotic treatment was administered quickly. Thus, we report a case in which genomic analysis was the critical factor in determining the best antimicrobial therapy for administration.