Metabolic profiling of serum for osteoarthritis biomarkers

Osteoarthritis is a prevalent aging disease in the world, and in recent years it has shown a trend toward younger age, which is becoming a major health problem in the world and seriously endangers the health of the elderly. However, the etiology and pathogenesis of osteoarthritis are still unclear, causing great trouble for treatment. To screen out potential biomarkers that could be used as identification of osteoarthritis and explore the pathogenesis of osteoarthritis, we performed untargeted metabolomics analysis of nine New Zealand rabbit serum samples by LC-MS / MS, including three normal serum samples (control group) and six osteoarthritis serum samples (case group). Finally 44 differential metabolites were identified, and the ROC analysis results indicated that a total of 36 differential metabolites could be used as potential biomarkers. Further metabolic pathway enrichment analysis was performed on these differential metabolites, and we found that a total of 17 metabolic pathways were affected, which may provide directions for the study of osteoarthritis mechanisms.

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

Rapid FRET-based homogeneous immunoassay of procalcitonin using matched carbon dots labels

A novel method for the detection of procalcitonin in a homogeneous system by matched carbon dots (CDs) labeled immunoprobes was proposed based on the principle of FRET and double antibody sandwich method. Blue-emitting carbon dots with a strong fluorescence emission range of 400-550nm and red-emitting carbon dots with the best excitation range of 410-550nm were prepared before they reacted with procalcitonin protoclone antibody pairs to form immunoprobes. According to the principles of FRET, blue-emitting carbon dots were selected as the energy donor and red-emitting carbon dots as the energy receptor. The external light source excitation (310nm) could only cause weak luminescence of CDs. However, once procalcitonin was added, procalcitonin and antibodies would be combined with each other quickly (≤ 20 min). Here, blue-emitting carbon dots acquired energy could be transferred to red-emitting carbon dots efficiently, causing the emitted fluorescence enhancement of red-emitting carbon dots. The fluorescence detection results in PBS buffer solution and diluted rabbit blood serum showed that the fluorescence intensity variation was linear with the concentration of procalcitonin. There was a good linear relationship between F/F0 and procalcitonin concentrations in PBS buffer solution that ranged from 0 to 100ng/ml, and the linear equation was F/F0 = 0.004 * Cpct + 0.98359. Detection in the diluted rabbit serum led to the results that were linear in two concentration ranges, including 0-40ng/ml and 40-100ng/ml, and the detection limit based on 3σ/K was 0.52ng/ml. It’s likely that this matched CDs labeled immunoprobes system can provide a new mode for rapid homogeneous detection of disease markers.

Elucidating the Structural and Minimal Protective Epitope of the Serogroup X Meningococcal Capsular Polysaccharide

Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment were further detailed at the atomic level by saturation transfer difference nuclear magnetic resonance (NMR) spectroscopy. The NMR results were used for validation of the in silico docking analysis between the X-ray crystal structure of the antibody (Fab fragment) and the modeled hexamer oligosaccharide. The antibody recognizes the MenX fragment by binding all six repeating units of the oligosaccharide via hydrogen bonding, salt bridges, and hydrophobic interactions. In vivo studies demonstrated that conjugates containing five to six repeating units can produce high functional antibody levels. These results provide an insight into the molecular basis of MenX vaccine-induced protection and highlight the requirements for the epitope-based vaccine design.

Identification and Expression Profiling of Circulating MicroRNAs in Serum of Cysticercus pisiformis-Infected Rabbits

Cysticercus pisiformis (C. pisiformis), the larval form of Taenia pisiformis, parasitize mainly the liver, omentum and mesentery of rabbits and cause huge economic losses in the rabbit breeding industry. MicroRNA (miRNA), a short non-coding RNA, is widely and stably distributed in the plasma and serum. Numerous data demonstrates that, after parasitic infection, miRNAs become the key regulatory factor for controlling host biological processes. However, the roles of serum miRNAs in C. Pisiformis-infected rabbits have not been elucidated. In this study, we compared miRNA expression profiles between the C. pisiformis-infected and healthy rabbit serum using RNA-seq. A total of 192 miRNAs were differentially expressed (fold change ≥ 2 and P < 0.05), including 79 up- and 113 downregulated miRNAs. These data were verified by qRT-PCR (real time quantitative polymerase chain reaction) analysis. Additionally, GO analysis showed that the target genes of these dysregulated miRNAs were most enriched in cellular, single-organism and metabolic processes. KEGG pathway analysis showed that these miRNAs target genes were involved in PI3K-Akt, viral carcinogenesis and B cell receptor signaling pathways. Interestingly, after aligning clean reads to the T. pisiformis genome, four (miR-124-3p_3, miR-124-3p_4, miR-124a and novel-miR1) T. pisiformis-derived miRNAs were found. Of these, novel-miR1was upregulated in different periods after C. pisiformis infection, which was verified qRT-PCR, and pre- novel-miR-1 was amplified from the cysticerci by RT-PCR, implying novel-miR-1 was derived from C. pisiformis and has great potential for the diagnosis of Cysticercosis pisiformis infection. This is the first investigation of miRNA expression profile and function in the serum of rabbits infected by C. pisiformis, providing fundamental data for developing diagnostic targets for Cysticercosis pisiformis.

Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

Effect of biotherapics made from serum or rabbit serum in Trypanosoma cruzi infection

The use of biotherapics as an intervention in experimental models of infection is a possible means to understand the effects of these medications [1-3]. This study evaluated the immunological and parasitological effects of biotherapics that were prepared from mouse or rabbit serum uninfected (MUI or RUI groups) or chronically infected with T. cruzi (MI or RI groups), dynamization 13cH. Male Swiss mice, 28 days of age were infected with T. cruzi-Y strain and treated with the different biotherapics diluted in water (1mL/100mL). The study was approved by the Ethics Committee for Experiments in Animals-UEM (protocol no. 080/2012). Using biotherapics made with mouse serum, MUI group exhibited good outcome with a pronounced Th1 response that was attributable to a reduction of IL-4 concentrations and decrease in IL-17 concentrations compared with the control group. However, this cytokine balance was not sufficient to promote decreased parasitemia in treated animals, likely because of a decrease in IFN-gama, thus hindering a more effective beneficial Th1 response. In contrast, the MI group presented a pronounced Th2 response that was attributable to increase in IL-4 and decrease in IFN-gama concentration compared to control group. MI group exhibited the worst outcome where the cytokine balance suppressed the immune response to T. cruzi in murine infection, resulting in a significant increased parasitemia and decreased survival time. Using biotherapics made with rabbit serum, RUI group exhibited the best outcome, including decreased parasitemia, with pronounced Th1 response that was attributable to decrease in IL-4 concentrations, with no changes in TNF-alpha and IFN-gama, associated to decrease in IL-17 compared to control group. In contrast, RI group did not exhibit alterations in parasitemia but a pronounced Th2 response that was attributable to increase in IL-4 concentration, with no changes in TNF-alpha and IFN-gama, associated to decrease in IL-17 compared to control group. Results show that biotherapics that were prepared from mouse or rabbit serum uninfected or chronically infected with T. cruzi differentially modulate the immune system in mice infected with this protozoan. Also, results provide evidence that biotherapics prepared with serum from healthy animal performed better than one made with serum from infected animal. In the same way biotherapics prepared with serum from resistant specie performed better than one made with serum from susceptible specie.

Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Development of rapid and cost-effective top-loading device for the detection of anti-SARS-CoV-2 IgG/IgM antibodies

Infection with SARS-CoV-2, the Betacoronavirus, caused a pandemic that affected the globe negatively. The gold method, RT-PCR, can detect SARS-CoV-2 but it is time-consuming and needs sophisticated equipment and professional personnel. On the other hand, rapid tests offer fast results and can detect anti-SARS-CoV-2 antibodies (Abs). The aim of this study is to develop a new rapid and cost-effective method for the detection of anti-SARS-CoV-2 IgG/IgM Abs. A new top-loading detection device was developed and composed of a small piece of plastic (25 × 25 × 0.5 mm) with an opening in the center, a piece of nitrocellulose (NC) membrane enough to block the opening from one side and adhesive tape to affix the NC to the plastic piece. The NC is blotted with anti-human IgG/IgM and rabbit serum. The device was evaluated against a commercially available IgG/IgM ELISA detection kit using normal, Covid-19-positive, HCV, HBV, and Cytomegalovirus-positive sera. Outcomes demonstrated simplicity, reproducibility, and accuracy of the new device and results can be obtained in less than 5 min. We anticipate our developed assay method to be used widely in point of care before deciding on the use of expensive nucleic acid assays.

Characterization of SARS-CoV-2 East Java isolate, Indonesia

Background: Incidents of SARS-CoV-2 in East Java increased steadily, and it became the second epicenter in Indonesia. The COVID-19 pandemic caused a dire multisectoral crisis all around the world. This study investigates and characterizes local isolates from East Java, Indonesia. Methods: There were 54 patients suspected with SARS-COV-2 infection and 27 patients were COVID-19 positive. Virus isolates were obtained from COVID-19 inpatients’ nasopharyngeal swabs at the Dr Soetomo Teaching Hospital, Surabaya. There were only three isolates (#6, #11, #35) with good growth characteristics. Serial blind passage and cytopathic effect observation in the Vero E6 cell line were performed for virus isolation. Confirmation of the SARS-CoV-2 infection was proven by means of reverse transcriptase-polymerase chain reactions using SARS-CoV-2 specific primers, scanning electron microscopy, and scanning transmission electron microscopy examination. Whole genome sequencing was performed using ARTIC protocol. Furthermore, SARS-CoV-2 characterization was identified through a western blot using rabbit serum immunized with inactive SARS-CoV-2 vaccine and human natural COVID-19 infection serum. Results: Spike gene analysis of three samples (#6, #11, #35) found that the D614G mutation was detected in all isolates, although one isolate exhibited the D215Y and E484D mutation. Based on whole genome analysis, those three isolates were included in clade 20A, and two isolates were included in lineage B.1.6 with one isolate belongs to lineage B.1.4.7. Conclusion: Based on molecular characterization and immunogenicity of SARS-CoV-2 East Java, Indonesia showed high titer and it has mutation in some regions.