Dot Blot Method Research Articles

NSUN2 methylates IRF4 to affect the capacity of macrophages attached to titanium implant on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs in vitro

BackgroundWe aimed to investigate the effect and underlying mechanism of titanium (Ti) implant on the polarization of macrophages and subsequent effects on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and angiogenesis of human umbilical vein endothelial cells (HUVECs) affected by macrophages.MethodsFirstly, the regulatory effect of Ti implant on macrophage polarization was investigated. Levels of M1 polarization markers and M2 polarization markers in macrophages were evaluated by immunofluorescence staining method and qPCR analysis. The osteogenic differentiation capacity of PDLSCs cultured with supernatants of macrophages of each group was evaluated by the Alizarin Red S (ARS) staining method and qPCR. Angiogenesis related genes were also evaluated in HUVECs cultured in supernatants of macrophages. To explore whether RNA m5C modification can modulate the effects of Ti implant on macrophage regulation of osteogenic differentiation and angiogenesis, we analyzed the main genes related to m5C in macrophages using RNA m5C dot blotting and qPCR methods. The interaction between NSUN2 and IRF4 was verified by m5C-RIP, RIP, and double-luciferase gene report experiments.ResultsMacrophages were activated as M1 macrophages under the interference of LPS, and macrophages attached to Ti implants were more easily activated as M2 macrophages under the action of LPS. Macrophages activated by Ti implant enhanced osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. NSUN2 level was up-regulated in macrophages treated with LPS and was down-regulated by Ti implant. Over-expression of NSUN2 attenuated the effect of Ti implant on M1 polarization promotion of macrophages and enhanced the M2 polarization promotion of macrophages. Up-regulation of NSUN2 weakened the effects of Ti implant on promotion the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs. The KEGG analysis suggested that IRF4 was enriched in several inflammatory signaling pathways. Moreover, NSUN2 methylates IRF4 to affect the capacity of macrophages on osteogenic differentiation of PDLSCs and angiogenesis of HUVECs.ConclusionsTaken together, macrophages of M1 type can be stimulated by Ti implants in vitro, promoting osteogenic differentiation of PDLSCs and angiogenesis in HUVECs through NSUN2-mediated methylation of IRF4.

A Dot-Blot Screening for Identifying the Temozolomide-Regulated Proteins as Potential Targets for Glioma Multi-OMICS Studies.

Malignant gliomas represent a heterogenous group of brain cancers that are characterized by infiltrative growth that lacks a clearly identifiable tumor border. The lack of the possibility of radical surgical resection and targeted therapy results in a poor prognosis. Although Temozolomide (TMZ) is still the leading chemotherapeutic agent in glioma treatment, its efficacy is limited due to the development of tumor resistance. Therefore, there an urgent need to improve the diagnosis and treatment of these tumors. Finding and developing biomarkers that are specific to glioma could be useful for both identifying therapy targets and monitoring treatment as well as for constructing a personalized therapy. However, there are still no reliable markers that would change the quality of glioma treatment. In this study, differences in the expression of 84 cancer-related proteins in three glioma cell lines were analyzed using the dot-blot method: commercial T98G cells and two patient-derived cell lines. The influence of TMZ on changes in protein expression, cell morphology, and migration was also investigated (Proteome Profiler Human XL Oncology Array, LeviCell System, Microscopic imaging). The lines that were analyzed were characterized by a remarkably different plasticity of protein expression and the proteomic alterations that were induced by TMZ. A dot-blot analysis revealed ten proteins that were common to all of the lines and five (Cathepsin b, FGF, Survivin, AXL, Osteopontin) that were modulated by the TMZ. As a result of the exposure of TMZ, the proteins that are involved in chemoresistance and invasion (TIE-2, Thrombospondin) were detected in both the HROG02 and T98G cell lines. In the control culture (not exposed to TMZ) of HROG17 cells, the proteins that are involved in metabolism were strongly suppressed. The presented data sheds new light on the modulatory effect of Temozolomide on the expression of a protein panel: Cathepsin b, fibroblast growth factor (FGF), Survivin, AXL, and Osteopontin that may suggest their potential as therapeutic targets or biomarkers to monitoring therapy effects. However, further high-throughput analysis and detection of the proteins in the body fluids are necessary.

ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF ETHYL ACETATE EXTRACT OF ACTINOMYCETES ISOLATED FROM TERMITE NESTS

Actinomycetes are an important source for the discovery of bioactive secondary metabolite compounds. Over 10,000 bioactive metabolite compounds have been isolated from terrestrial actinomycetes with various biological activities, such as antibiotic, antiviral, anti-inflammatory, antitumor, anticancer, and antioxidant. One source of origin for actinomycetes is soil, including termite nests. Utilization of natural sources such as actinomycetes from termite nests as antioxidants and antibacterials can be an alternative source of production of new secondary metabolite compounds. This research aims to evaluate the antioxidant and antibacterial activities of actinomycete extracts from termite nests. Antioxidant activity using the DPPH free radical scavenging method, and antibacterial activity against S. aureus. Antioxidant and antibacterial activity were performed by using the TLC dot-blot, TLC bioautography, and microdilution methods to determine the inhibitory concentration of 50% (IC50), antioxidant activity index (AAI), and minimum inhibitory concentration (MIC). From the 33 extracts tested, 16 extracts showed antioxidant activity with IC50 values range of 76.64-126.22 µg/mL or AAI value > 0.05 (moderate), and 8 extracts had moderate antibacterial activity against S. aureus (MIC values range of 256-512 µg/mL). Future research for scaling-up of actinomycete culture, isolating of active compounds, determining of the chemical structure of active compounds, and further testing as antioxidants and antibacterials still need to be carried out.

The Blood Group Rhc Protein from Human Erythrocyte Membranes as an Immunogen for Producing Antibodies in Mice

Background: Various methods have been used to isolate red blood cell (RBC) membrane antigens. In this regard, obtaining the antigen and preserving its structure is of special importance. However, limited studies have been conducted to purify cellular membrane antigens such as Rh proteins.
 Materials and Methods: In this experimental study, Rhc antigens of the RBC membrane was purified. Here, the RBC membrane was solubilized through the lysis buffer. Next, dialysis and affinity chromatography were performed using polyclonal anti-human RhCcEe antibody to isolate Rhc/e antigens from the RBCs with the following blood group characteristics: Rhc+, RhC-, Rhe+, and RhE-. The purified proteins were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and dot blot methods. The immunization process was performed in Balb/c mice using the Rhc antigen as an immunogen. After the last injection, the mouse serum was used to titrate antibodies.
 Results: Protein bands of the purified antigen were observed in the silver-stained SDS-PAGE gel (region of 25-35 kDa). The OD405nm = 0.56 ± 0.05 results showed the reactivity with Rhc antibody. The specificity of the purified protein was evaluated using the dot blot assay. The anti-sera titration was greater than 1/10,000 against Rhc-coated microwells. Rh antigens can be isolated from the RBC membrane using the non-ionic NP-40 detergent and affinity chromatography.
 Conclusion: The Rh antigen can be isolated from the RBC membrane with proper purity by solubilization with the non-ionic NP-40 detergent and purification by affinity chromatography. It seems that the membrane antigen maintains its antigenicity and structure. As a result, it can be detected by blood group-specific antibodies used in the hemagglutination method. Purified antigens may be used to generate antibodies or to study the protein structure.

HPV positivity status in males is related to the acquisition of HPV infection in females in heterosexual couples

PurposeFew studies have focused on the impact of human papillomavirus (HPV) positivity in male partners on female HPV infection and cervical lesions. The purpose of this study was to evaluate the impact of the HPV infection status of husbands on wives’ cervical HPV infection and lesions.MethodsWe surveyed 251 monogamous couples who attended the outpatient department of Fujian Maternity and Child Health Hospital from 2013 to 2021. HPV type analysis was performed on exfoliated cells of the females’ cervix and males’ urethra by the PCR-reverse dot blot method. We analyzed the prevalence and consistency of HPV types in 251 couples. Subsequently, the risk of HPV infection in females with HPV-positive male partners was analyzed. SPSS version 26 (IBM, Chicago, USA) was used for statistical analysis.ResultsIn 251 couples, the most commonly detected high-risk HPV (HR-HPV) genotypes were 52, 51, 16, and 58 for males and 16, 52, 18, and 58 for females. Wives with HPV-positive husbands had higher infection rates for most HR-HPV genotypes. HR-HPV positivity in husbands was a risk factor for the development of cervical lesions in wives (OR = 2.250, P = 0.014). Both single-type (OR = 2.085, P = 0.040) and multiple-type (OR = 2.751, P = 0.036) infection in husbands will contributed to an increased risk of non-HR-HPV infection and cervical lesions in wives.ConclusionHusbands’ HPV positivity increases the burden of non-HR-HPV infection and increases the risk of cervical lesions developing in wives. It is hoped to provide a reference value for cervical cancer prevention in females and HPV vaccination in males.

318P Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined for detecting metastases in sentinel lymph nodes of patients with breast cancer: A multi-center prospective study

318P Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined for detecting metastases in sentinel lymph nodes of patients with breast cancer: A multi-center prospective study

Spray losses study of two pesticides by UASS in integrated rice-crayfish farming system and acute toxicity evaluation on Procambarus clarkii.

While the integrated rice-crayfish (Procambarus clarkii) farming system (IRCFS) is widely developing in China, the widespread use of Unmanned Aerial Spraying Systems (UASS) to protect rice from pests has led to potential pesticide risk for the crayfish in IRCFS. Therefore, it is crucial to examine UASS's spray deposition and drift in IRCFS. In this study, we used the oligonucleotide sequence-tracking / dot-blotting (OSTDB) method to trace pesticide spraying. We collected detailed data not only on spray loss in the paddy fields, but also on spray drift in the breeding ditches caused by upwind and downwind spray areas. Additionally, pesticide residues in the breeding ditches were measured using LC-MS/MS by collecting water samples after pesticide application. The data analysis indicated that the spray loss in the paddy field was significantly greater than that in the breeding ditches. The spray drift in the breeding ditches, caused by the upwind spray area, was seven times higher than that originating from the downwind spray area. Furthermore, the results also revealed that the bulk flow between the paddy fields and the breeding ditches contributed a substantial amount of pesticide residue to the water body in the breeding ditches. In addition, we investigated the acute toxicities of common insecticides using in paddy fields, including thiamethoxam (THI), chlorantraniliprole (CHI), THI·CHI-Mix and THI·CHI-WG. The results demonstrated that the spray losses and spray drift from UASS spray applications of these pesticides in IRCFS would not cause acute toxicity or death in crayfish. These findings provide important materials for establishing pesticide application standards and guiding the field testing of droplet deposition and drift in IRCFS.

Bio-Membrane SELEX as a New Approach for Selecting ss-DNA Aptamers that Bind to the Hydatid Cyst Laminated Layer.

Hydatid cyst (HC) is the larval stage of the canine intestinal tapeworm (cestode), Echinococcus granulosus. In addition to the high global economic cost of livestock farming, the infection can lead to dangerous problems for human health. Therefore, research into new diagnosis and treatment approaches is valuable. This study is set out to explore aptamers that bind to HC antigens. The similarity between HC genotype in sheep and humans was that sheep HCs were collected and used as a biological membrane for aptamer selection. Four Bio- Membrane SELEX rounds were conducted, and ssDNA aptamers were selected. Selected aptamers' affinity and specificity to the laminated layer antigens were evaluated using membrane staining by fluorescein primer as a probe. Biotinylated primer was used as a probe for aptahistochemistry and dot blot techniques. Subsequently, cloning and plasmid extraction was conducted. The affinity and specificity of sequenced aptamers were examined with the dot blot method. Selected aptamers reacted with HC wall in aptahistochemistry, aptahistofluorescent, and dot blot experiments. Following cloning and sequencing, 20 sequences were achieved. A strong reaction between HC total antigens and sequenced aptamers has emerged in the dot blot method. We propose a novel method to determine specific aptamers in this investigation. Bio-Membrane SELEX could be assumed as a practical and sensitive method for aptamer selection. Selected aptamers in this study possibly may be used for specific HC antigens detection.

Hematological and molecular characteristics of a novel α-globin variant Hb Liangqing (HBA2:c.224A>G)

ABSTRACTObjectivesTo report the hematological and molecular characteristics of a novel α-globin variant found among Chinese families.MethodsThis study was done on two unrelated families (F1 and F2). Hematological results were obtained by an automated blood cell analyzer. Hemoglobin (Hb) fraction analysis was carried out using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Gap-PCR and reverse dot blot (RDB) methods were used to detect the common α-thalassemia mutations in the Chinese population. The Hb variants were defined by Sanger sequencing.ResultsHb fraction analysis of F2 cord blood using HPLC showed an abnormal peak (3.5%) of the S-window, but CE presented a 12.2% abnormal peak at zone 5(S). Similar results of CE were observed from the F1 twin's cord blood. Compared with newborns, Hb analysis of F2 father using HPLC demonstrated an abnormal peak (16.9%) of S-window and an unknown peak (0.5%) at a retention time of 4.60 min, respectively. In contrast, CE revealed a high Hb F peak at zone 7 and an unknown peak at zone 1. There was no abnormality detected with Gap-PCR and RDB in these patients. However, Sanger sequencing confirmed the presence of a new heterozygous mutation (GAC>GGC) at codon 74 of the HBA2 gene (HBA2:c.224A>G), resulting in a novel Hb variant. We named it Hb Liangqing for the birthplace of the proband.ConclusionThis is the first report of Hb Liangqing detected by HPLC and CE. The normal hematological phenotype suggests that it may be a benign Hb variant.

Shark-derived single-domain antibodies for the detection of decapod iridescent virus 1

Decapod iridescent virus 1 (DIV1) has emerged as a novel threat to shrimp farming. The development of a rapid, convenient, and accurate method for diagnosing DIV1 is urgently needed, serving as a key factor in the reduction of the risk of viral outbreaks. This study aimed to develop a new variable antigen receptor (vNAR), derived from shark heavy-chain antibodies, directed against DIV1 for use in detection assays. White-spotted bamboo sharks were immunized with DIV1 virions, and an immune phage-display library was constructed. After three rounds of panning, the lead D02 vNAR was isolated from the library. D02 vNAR was identified to target the viral antigen ORF064L through western blotting, LM-SC/SC, and pull-down analyses. Furthermore, indirect enzyme linked immunosorbent assay (indirect ELISA) and dot-blot assays were developed based on D02 vNAR. The detection limits of the indirect ELISA and dot blot methods were 5 ng/mL and 6.25 ng/spot, respectively. Furthermore, in the indirect ELISA method, a standard curve describing the log concentration of DIV1 versus OD value was established, which was linear in the concentration range of 80–10,240 ng/mL, and the linear regression equation was derived as y = 0.3565x − 0.51601. Furthermore, 300 clinical samples were analyzed by the two methods using quantitative real-time polymerase chain reaction (qRT-PCR) as a reference. Compared to qRT-PCR, the diagnostic sensitivity and specificity of the indirect ELISA and dot blot methods were 96.7 % and 100 % and 93.5 % and 100 %, respectively, demonstrating the practicability and reliability of the vNAR-based indirect ELISA and dot blot methods for the robust detection of DIV1. Overall, this is the first study in which a vNAR has been developed against DIV1. The developed vNAR offers a robust tool with great potential for the diagnosis of DIV1 during shrimp cultivation.

Serum-derived bovine immunoglobulin/protein isolate binds to various opportunistic microbes along with associated virulence factors

The gut microbiome consists of a complex population of bacteria, fungi, and viruses that aid the host in various physiological functions. When the microbiota is imbalanced, known as dysbiosis, symbiotic microbes can become opportunistic and excrete virulence factors that decrease intestinal barrier function, causing chronic inflammation that leads to disease. Therefore, maintaining gut microbiome balance is critical to overall health. Endogenous immunoglobulins play a positive role in gut homeostasis and immunoglobulin supplements can assist the body with microbiome maintenance. Serum-derived bovine immunoglobulin/protein isolate (SBI), a spray-dried protein powder enriched in immunoglobulins (IgG, IgA, IgM), is clinically proven to help manage loose and frequent stools associated with gastrointestinal enteropathies. A previous study used an in vitro cell culture model to show that SBI binds and neutralizes pattern-associated molecular patterns by steric/immune exclusion mechanisms. The goal of our study is to expand the list of antigens to which SBI binds. We hypothesize the IgG in SBI binds additional opportunistic microbes found in the gut and their associated virulence factors. Heliobacter pylori ( H. pylori), Shigella dysenteriae, Escherichia coli ( E. coli), and Candida albicans ( C. albicans) are common microbes associated with human gastroenteropathies. Therefore, we initially tested the binding of SBI to H. pylori lysate, H. pylori cytotoxicity-associated immunodominant antigen (CagA), Shiga toxin type-1 (Stx1), E. coli cytolethal distending toxin (CDT), C. albicans lysate, and C. albicans agglutinin-like protein 3 (Als3) by a dot blot method. SBI demonstrated binding to all antigens except the H. pylori lysate. To validate specificity of IgG, further binding studies were completed using a modified ELISA. The absorbance of IgG bound to immobilized antigens increased proportionally to SBI concentration (CagA R 2 =0.98, Stx1 R 2 =0.97, CDT subunit A R 2 =0.99, CDT subunit C R 2 =0.99, C. albicans lysate R 2 =0.99, and Als3 R 2 =0.99). Control experiments verified effective blocking, non-specific binding, and functionality (denatured SBI), as no significant absorbance was measured for any antigen tested. Together, the dot blot and modified ELISA showed the IgG in SBI was found to bind to a variety of opportunistic microbes and their associated virulence factors, including CagA, Stx1, CDT subunit A, CDT subunit C, C. albicans lysate, and Als3. Future work will examine if IgG binding can promote gut microbiome balance. Proliant Health and Biologicals This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of the semi-dry dot-blot method for intraoperative detecting micropapillary component in lung adenocarcinoma based on proteomics analysis.

Micropapillary (MIP) component was a major concern in determining surgical strategy in lung adenocarcinoma (LUAD). We sought to develop a novel method for detecting MIP component during surgery. Differentially expressed proteins between MIP-positive and MIP-negative LUAD were identified through proteomics analysis. The semi-dry dot-blot (SDB) method which visualises the targeted protein was developed to detect MIP component. Cellular retinoic acid-binding protein 2 (CRABP2) was significantly upregulated in MIP-positive LUAD (P < 0.001), and the high CRABP2 expression zone showed spatial consistency with MIP component. CRABP2 expression was also associated with decreased recurrence-free survival (P < 0.001). In the prospective cohort, the accuracy and sensitivity of detecting MIP component using SDB method by visualising CRABP2 were 82.2% and 72.7%, which were comparable to these of pathologist. Pathologist with the aid of SDB method would improve greatly in diagnostic accuracy (86.4%) and sensitivity (78.2%). In patients with minor MIP component (≤5%), the sensitivity of SDB method (63.6%) was significantly higher than pathologist (45.4%). Intraoperative examination of CRABP2 using SDB method to detect MIP component reached comparable performance to pathologist, and SDB method had notable superiority than pathologist in detecting minor MIP component.

ELISA-like QDB method to meet the emerging need of Her2 assessment for breast cancer patients.

Inherent issues of subjectivity and inconsistency have long plagued immunohistochemistry (IHC)-based Her2 assessment, leading to the repeated issuance of guidelines by the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) for its standardization for breast cancer patients. Yet, all these efforts may prove insufficient with the advent of Trastuzumab deruxtecan (T-Dxd), a drug with the promise to expand to tumors traditionally defined as Her2 negative (Her2-). In this study, we attempted to address these issues by exploring an ELISA-like quantitative dot blot (QDB) method as an alternative to IHC. The QDB method has been used to measure multiple protein biomarkers including ER, PR, Ki67, and cyclin D1 in breast cancer specimens. Using an independent cohort (cohort 2) of breast cancer formalin-fixed paraffin-embedded (FFPE) specimens, we validated cutoffs developed in cohort 1 (Yu etal., Scientific Reports 2020 10:10502) with overall 100% specificity (95% CI: 100-100) and 97.56% sensitivity (95% CI: 92.68-100) in cohort 2 against standard practice with the dichotomized absolutely quantitated values. Using the limit of detection (LOD) of the QDB method as the putative cutoff point, tumors with no Her2 expression were identified with the number comparable to those of IHC 0. Our results support further evaluation of the QDB method as an alternative to IHC to meet the emerging need of identifying tumors with low Her2 expression (Her2-low) in daily clinical practice.

Abstract P5-01-03: Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined with an automatic reader for detecting metastases in sentinel lymph nodes of patients with breast cancer: a multi-center prospective study

Abstract Background: The semi-dry dot-blot (SDB) method, a diagnostic procedure for detecting lymph node (LN) metastases using an anti-cytokeratin (CK) antibody, is based on the theory that epithelial components, such as CK, are not found in normal LNs. Thus, metastases are diagnosed according to the presence of CK in the lavage fluid of sectioned LNs. We prospectively evaluated novel SDB kits that use a newly developed anti-CK19 antibody and an automatic reader for diagnosing sentinel LN metastases in patients with breast cancer as a multi-center study. Methods: We obtained 924 sentinel LNs dissected from 405 patients with breast cancer between January 2021 and December 2021 at six institutes in Japan. We excluded patients who underwent neoadjuvant chemotherapy and neoadjuvant endocrine therapy. LNs were sectioned at 2-mm intervals and washed with phosphate-buffered saline. Cells suspended in the lavage fluid of sectioned LNs were centrifuged and lysed to extract protein. The extracted protein was applied to the SDB kit to diagnose LN metastasis using an automatic absorbance reader. Hematoxylin and eosin (H&amp;E) stained and washed LNs were blindly examined by pathologists using intraoperative and permanent histological examination. Diagnoses based on SDB kit and automatic reader findings were compared with diagnoses made by permanent histological examination of paraffin-embedded H&amp;E-stained sections of LNs. Primary endpoints were the sensitivity, specificity, and overall agreement of the SDB kit for distinguishing macrometastases from non-macrometastases. Results: Ninety-four of the 924 LNs were assessed as macrometastases, 40 as micrometastases, and 790 as isolated tumor cells by histological examination. Compared with patients with non-macrometastases, those with macrometastases had significantly younger age (p&amp;lt; 0.01), larger primary tumor (p&amp;lt; 0.01), higher nuclear grade (p=0.04), increased lymphatic invasion (p&amp;lt; 0.01), and increased venous invasion (p=0.01). Using a borderline CK19 absorbance of 11.9 milli-absorbance for detecting macrometastases with an area under the curve of 0.989, the sensitivity, specificity, and overall agreement of the SDB kit were 94.7%, 98.3%, and 97.9%, respectively. Moreover, the sensitivity, specificity, and overall agreement of the intraoperative histological examination compared with permanent histological examination for distinguishing macrometastases from non-macrometastases were 91.4%, 99.1%, and 98.3%, respectively. Furthermore, the kits and automatic reader yielded diagnoses within approximately 20 min at a cost of &amp;lt; 30 USD for the SDB kit and &amp;lt; 3,000 USD for the automatic reader. Conclusions: The kits with an automatic reader used in our study were accurate, quick, and cost-effective in diagnosing LN metastases without the loss of LN tissue and were particularly useful for distinguishing macrometastases. We plan to make the SDB kit and automatic reader commercially available worldwide soon. Citation Format: Megumi Matsumoto, Masaaki Baba, Aya Tanaka, Sayaka Kuba, Michi Morita, Fujiko Kaseida, Hiroko Hayashi, Shigeto Maeda, Hiroshi Yano, Eiko Inamasu, Akiko Ogiya, Kenta Sekiya, Masahiro Nakashima, Hideki Ikari, Shinji Ohno, Hiroyuki Takei, Katsunori Yanagihara, Susumu Eguchi, Takeshi Nagayasu, Ryota Otsubo. Evaluation of novel diagnostic kits using the semi-dry dot-blot method combined with an automatic reader for detecting metastases in sentinel lymph nodes of patients with breast cancer: a multi-center prospective study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-01-03.

Promotion of the resistance of human oral epithelial cells to herpes simplex virus type I infection via N6-methyladenosine modification

ObjectiveThis study aimed to explore the mechanism behind N6-methyladenosine (m6A) modification of the total ribonucleic acid (RNA) involved in the resistance to herpes simplex virus type I (HSV-1) infection in oral epithelial cells.MethodThe variation in m6A modification level on messenger RNA following HSV-1 infection was determined using the RNA dot blot method. The expression levels of alpha-ketoglutarate-dependent dioxygenase lab homolog 5 (ALKBH5) protein and fatty mass and obesity-associated genes (FTO) were determined using real-time fluorescence quantification polymerase chain reaction and the western blot technique, respectively. Next, after suppressing the expression of ALKBH5 or FTO via small interfering RNA, human immortalised oral epithelial cells (HIOECs) were infected with HSV-1, followed by measurement of the viral load or expression level of type I interferon (I-IFN) and interferon-stimulated genes (ISGs).ResultsThe m6A modification level was significantly increased following HSV-1 infection of the HIOECs (P < 0.05), while the expression of ALKBH5 and FTO genes was reduced (P < 0.01). Moreover, the suppression of ALKBH5 or FTO increased the production of I-IFN and ISGs during the HSV-1 infection of the HIOECs (P < 0.01), and the viral load was significantly reduced (P < 0.01).ConclusionDuring oral HSV-1 infection, the m6A level was increased through the down-regulation of ALBHK5 and FTO expression, increasing I-IFN production and the promotion of HSV-1 clearing in HIOECs.

Hypercitrullination and anti-citrullinated protein antibodies in chronic apical periodontitis, a laboratory investigation. Does autoimmunity contribute to the pathogenesis?

The presence of Gram-negative anaerobic bacteria, in particular, Porphyromonas gingivalis (P.gingivalis) in periapical granulomas predicts the generation of citrullinated proteins in the lesion. Citrullination of proteins may lead to the formation of anti-citrullinated autoantibodies (ACPA-s) initiating the formation of an autoimmune loop which may contribute to the perpetuation of inflammatory reactions and tissue damage in chronic apical periodontitis. The objective of this study was to demonstrate the formation of citrullinated proteins in chronic apical periodontitis and whether they can act as autoantigens. Twenty-five periapical granulomas (n=25) were investigated in the study. Healthy periodontal tissue samples served as normal control tissue (n=6). The peptidyl-citrulline level was determined with the dot blot method. ACPA levels were analysed using anti-citrullinated cyclic peptide (anti-CCP) EDIA kit. Differences between periapical granuloma and control samples were assessed using Mann-Whitney U tests. p Values <.05 were considered as statistically significant. Protein concentrations, peptidyl-citrulline levels and anti-CCP ratios were compared between periapical granuloma and healthy control groups. Multiple linear regression analysis revealed significant (p=.042) hypercitrullination in periapical granuloma samples. Moreover, there was a significant difference in the ACPA ratios between periapical granuloma (2.03 ± 0.30) and healthy control (0.63 ± 0.17) groups (p=.01). Seventeen of 25 periapical granuloma samples (17/25; 68%), whereas one out of six control samples (1/6; 17%) were shown to be positive for the presence of ACPA. This is the first study detecting the presence of citrullinated peptides and APCA in periapical granuloma, suggesting the contribution of autoimmune reactions in the pathogenesis and perpetuation of chronic apical periodontitis.

Cell-Free Dot Blot: an Ultra-Low-Cost and Practical Immunoassay Platform for Detection of Anti-SARS-CoV-2 Antibodies in Human and Animal Sera.

Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.

THRIFTY-A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection.

Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type-dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type-isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology. Graphical Overview Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).

Selection and characterization of a new human Interleukin-17A blocking DNA aptamer using protein-SELEX

BackgroundInterleukin-17A (IL-17A) is an important pro-inflammatory cytokine observed in the development of many disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis. The anti-IL-17A biological drugs, including Secukinumab, Ixekizumab, and Brodalumab, are monoclonal antibodies approved for several disease treatments. Due to the disadvantages of biological therapies, including their immunogenicity, difficulties in scale generation, and high production costs and time, it is necessary to find new alternative anti- IL-17A agents for these monoclonal antibodies. Our study aimed to identify ssDNA aptamers that block IL-17A activity using the protein-SELEX procedure. MethodsThe hIL-17A was expressed in codon plus E. coli, and after 14 rounds of the SELEX process, monitoring of aptamer pools was done using the dot blot method. Three families of aptamers were obtained from the selected round 9 aptamer pool, and seven truncates were created. Inhibitory effects of aptamer truncate on IL-17-induced CCL20 expression in HaCaT keratinocytes were evaluated. ResultsAll aptamer truncates had a significant inhibitory effect compared to the library, but the inhibitory effect of M2 and M7 truncates was more than 80%. Moreover, we evaluated the potential binding site of selected aptamers by ELISA. ConclusionsWe introduced a new small 17-nucleotide DNA aptamer that efficiently binds and blocks hIL-17A with a 0.3 nM kd, a potential anti-IL-17A therapeutic agent.

Investigação de Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. e Alphacoronavirus sp. em tamanduás-bandeira (Myrmecophaga tridactyla) em cativeiro

ABSTRACT This research aimed to investigate the occurrence of Chlamydia sp., Morbillivirus sp., Parvovirus sp., Leishmania sp. and Alphacoronavirus sp. in captive giant anteaters. Blood and fecal samples were taken from 16 animals in institutions from the states of Minas Gerais, Bahia and Distrito Federal, which had been in captivity for at least a year. A commercial rapid chromatographic immunoassay test was used for detecting coronavirus and parvovirus antigens, in addition to antibodies against leishmaniasis, all results being negative. In the case of the test for antibodies against distemper, four (4/16; 25%) anteaters had an average titration, two (2/16; 12.5%) a low titration and ten (10/16; 62.5%) were non-reactive. Using the DOT-ELISA (dot blotting) method for detection of immunoglobulin G, only one specimen obtained a 1 : 40 titration. For the polymerase chain reaction tests for Leishmania and Chlamydia, all samples were negative.